Methods and compositions for treatment of muscle wasting, muscle weakness, and/or cachexia

ABSTRACT

Embodiments of the invention include methods of treating, preventing, and/or reduce the risk or severity of a condition selected from the group consisting of muscle wasting, muscle weakness, cachexia, and a combination thereof in an individual in need thereof. In some embodiments, particular small molecules are employed for treatment, prevention, and/or reduction in the risk of muscle wasting. In at least particular cases, the small molecules are inhibitors of STAT3.

This application is a continuation application of U.S. Non-Provisionalapplication Ser. No. 14/335,853 filed Jul. 18, 2014 which claimspriority to U.S. Provisional Patent Application Ser. No. 61/847,778filed Jul. 18, 2013, both of which are incorporated by reference hereinin their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 CA058183, K08HL085018-01A2, P50 CA097007, R21 CA149783, and R41 CA153658, awarded byNational Institutes of Health. The United States Government has certainrights in the invention.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing, named“SeqLst_BAYM_P0112USC1_1001121419_BLG_14_005.txt” (4,602 bytes) whichhas been submitted electronically in ASCII format and is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention generally concerns at least the fields of cellbiology, molecular biology, and medicine.

BACKGROUND OF THE INVENTION

Muscle wasting is a debilitating complication of catabolic conditionsincluding chronic kidney disease (CKD), diabetes, cancer or seriousinfections. Unfortunately, there are few reliable strategies that blockthe loss of muscle protein initiated by these conditions. Previously, itwas found that myostatin, a negative regulator of muscle growth, isincreased in muscles of mice with CKD and when myostatin is inhibitedwith a “humanized” myostatin peptibody, CKD-induced muscle wasting wasblocked (Zhang et al., 2011a). A similar conclusion was reached instudies of mouse models of cancer cachexia (Zhou et al., 2010). In themice with CKD, inhibition of myostatin reduced circulating levels ofIL-6 and TNFα suggesting a link between inflammation and muscle wastingas reported in clinical studies (Carrero et al., 2008; Hung et al.,2011). The evidence that inflammation stimulates muscle wasting includesreports that infusion of TNFα, IL-6, IL-10 or IFN-γ into rodents resultsin muscle wasting while neutralization of cytokines using genetic orpharmacological approaches attenuates muscle wasting (Cheung et al.,2010). For example, rodents were treated with a constant infusion ofangiotensin II (AngII) and found there was muscle wasting plus increasedcirculating levels of IL-6 and increased expression of SOCS3 withsuppressed insulin/IGF-1 signaling; knockout IL-6 from mice suppressedAng II induced muscle wasting (Zhang et al., 2009; Rui et al., 2004; Ruiet al., 2002).

Responses to IL-6 or INFγ involve stimulation of intracellular signalingpathways including activation of Janus protein tyrosine kinases (JAKs).Subsequently, JAKs mediate tyrosine phosphorylation of Signal Transducerand Activator of Transcription (STAT) factors followed by theirdimerization, nuclear translocation and activation of target genes(Horvath, 2004). Among the seven members of the Stat family, Stat3 isthe major member that is activated by the IL-6 family of cytokines(Hirano et al., 1997; Kishimoto et al., 1994). Recently, Bonetto et alreported the results of a microarray analysis of muscles from mice withcancer-induced cachexia. Components of 20 signaling pathways wereupregulated, including IL-6, Stat3, JAK-STAT, SOCS3, complement andcoagulation pathways. Therefore, the Stat3 pathway could be linked toloss of muscle mass but the pathway from Stat3 to muscle wasting isunknown.

A potential target of activated Stat3 is C/EBPδ. The C/EBP transcriptionfactors (C/EBP-α, -β, -γ, -δ, -ω, and -ζ) are expressed in severaltissues and act to regulate inflammatory and metabolic processes (Ramjiand Foka, 2002). C/EBP-β or -δ can stimulate intracellular signaling inhepatocytes or inflammatory cells (Poli, 1998; Akira et al., 1990;Alonzi et al., 1997) and in mice responding to an excess ofglucocorticoids, the expression and binding activity of C/EBP-β and -δin muscle are increased (Penner et al., 2002; Yang et al., 2005).

One embodiment that includes C/EBPδ involves increased myostatinexpression because the myostatin promoter contains recognition sites forglucocorticoid receptors, forkhead transcription factors as well asmembers of the C/EBP family of transcription factors (Ma et al., 2003;Allen and Unterman, 2007). In the present disclosure, an intracellularsignaling pathway in cultured myotubes is identified that bridges thegaps between p-Stat3 and myostatin and loss of muscle mass. To examineif the pathway was operative in vivo, it was studied how two catabolicconditions, CKD or acute, streptozotocin-induced diabetes affect musclemetabolism in a muscle-specific Stat3 knockout (KO) mouse. It was alsotested whether a small molecule inhibitor of Stat3 phosphorylation wouldcorrect muscle wasting. Interruption of Stat3 improved muscle metabolismand strength in mice with CKD and evidence was gathered for the pathwayin muscle biopsies from patients with CKD.

The present disclosure satisfies a need in the art to provide novelcompounds and methods for treating and/or preventing muscle wasting orcachexia in individuals.

SUMMARY OF THE INVENTION

Embodiments of the invention include methods and/or compositions for thetreatment of at least muscle wasting (which may occur as weakening,shrinking, and/or loss of muscle caused by disease, age, or lack of use)and/or muscle weakness and/or cachexia. The muscle wasting and/orweakness may be related to any underlying medical condition and be theresult of any cause. The underlying condition may or may not be known.In specific embodiments, the muscle wasting and/or muscle weakness maybe part of cachexia, and cachexia may also be treated with methods andcompositions of the invention.

Embodiments of the invention include methods and/or compositions for thetreatment of muscle weakness and/or muscle wasting and/or cachexia in anindividual known to have the muscle weakness and/or muscle wastingand/or cachexia, suspected of having muscle weakness and/or musclewasting and/or cachexia, or at risk for having muscle weakness and/ormuscle wasting and/or cachexia. The compositions include small moleculesand functional derivatives as described herein. In some embodiments, theindividual is receiving an additional therapy for an underlyingcondition that is related to (and may be the direct or indirect causeof) the muscle weakness and/or muscle wasting and/or cachexia and/or theindividual is receiving an additional therapy for the muscle weaknessand/or muscle wasting and/or cachexia itself.

In embodiments of the invention, an individual is given more than onedose of one or more compositions described herein or functionalderivatives thereof. The dosing regimen may be separated in time byminutes, hours, days, months or years.

An individual in need thereof is an individual that has at least onesymptom of a condition selected from muscle weakness, muscle wasting andcachexia or any combination thereof or is susceptible to having acondition selected from muscle weakness, muscle wasting and cachexia orany combination thereof by having an underlying condition that can havea condition selected from muscle weakness, muscle wasting and cachexiaor any combination thereof as part of the underlying condition or as asecondary component of the underlying condition, for example.

Delivery of the composition of the invention may occur by any suitableroute, including systemic or local, although in specific embodiments,the delivery route is oral, intravenous, topical, subcutaneous,intraarterial, intraperitoneal, buccal, by aerosol, by inhalation, andso forth, for example.

In some embodiments of the invention, the methods and/or compositions ofthe invention are useful for treating and/or preventing and/or reducingthe risk of a condition selected from muscle weakness, muscle wastingand cachexia or any combination thereof, and in specific cases suchtreatment occurs by inhibiting Stat3 and/or Stat1 activity. In certainembodiments, the compositions inhibit Stat3 but fail to inhibit Stat1.In particular embodiments, the compositions do not inhibit Stat3 orStat1. In some embodiments, compounds of the invention interact with theStat3 SH2 domain, competitively inhibit recombinant Stat3 binding to itsimmobilized pY-peptide ligand, and/or inhibit IL-6-mediated tyrosinephosphorylation of Stat3, for example. In particular embodiments, thecompositions of the invention fulfills the criteria of interactionanalysis (CIA): 1) global minimum energy score ≤−30; 2) formation of asalt-bridge and/or H-bond network within the pY-residue binding site ofStat3; and/or 3) formation of a H-bond with or blocking access to theamide hydrogen of E638 of Stat3, for example. In some embodiments, thecomposition(s) interacts with a hydrophobic binding pocket with theStat3 SH2 domain.

In a specific embodiment of the invention, there is a method oftreating, preventing, and/or reducing the risk of a condition selectedfrom muscle weakness, muscle wasting and cachexia or any combinationthereof in an individual comprising delivering to the individual atherapeutically effective amount of a compound selected from the groupconsisting ofN-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide,a functionally active derivative thereof, and a mixture thereof.

In a specific embodiment of the invention, there is a method oftreating, preventing, and/or reducing the risk of a condition selectedfrom muscle weakness, muscle wasting and cachexia or any combinationthereof in an individual comprising delivering to the individual atherapeutically effective amount of a compound selected from the groupconsisting of4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoicacid;4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoicacid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid;3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid; methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate;4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoicacid; a functionally active derivative thereof; and a mixture thereof.In a specific embodiment, any of the compounds disclosed herein aresuitable to treat and/or prevent cachexia, for example.

In another embodiment, the inhibitor comprises the general formula:

wherein R₁ and R₂ may be the same or different and are selected from thegroup consisting of hydrogen, carbon, sulfur, nitrogen, oxygen,flourine, chlorine, bromine, iodine, alkanes. cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, and benzoic acid-basedderivatives.

In another embodiment of the invention, the composition comprises thegeneral formula:

wherein R₁, and R₃ may be the same or different and are selected fromthe group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen,flouring, chlorine, bromine, iodine, alkanes. cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, and benzoic acid-basedderivatives; and R₂ and R₄ may be the same or different and are selectedfrom the group consisting of hydrogen, alkanes, cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, and benzoic acid-basedderivatives.

In another embodiment of the invention, the composition comprises thegeneral formula:

wherein R₁, R₂, and R₃ may be the same or different and are selectedfrom the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen,fluorine, chlorine, bromine, iodine, carboxyl, alkanes. cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, and benzoic acid-basedderivatives.

In specific embodiments, the condition selected from muscle weakness,muscle wasting and cachexia or any combination thereof treated by thecomposition may be in an individual with any type of cancer. In somecases, the cancer may be of the lung, breast, skin, liver, kidney,testes, ovary, cervix, bone, spleen, gall bladder, brain, pancreas,stomach, anus, prostate, colon, blood, head and neck, or lymphoidorgans. For example; the composition may inhibit Stat3 in a cell of themuscles or other tissues of individuals with any of these cancers.Mammals may be treated with the methods and/or compositions of theinvention, including humans, dogs, cats, horses, cows, pigs, sheep, andgoats, for example.

In other embodiments of the invention, there are methods of treating acondition selected from muscle weakness, muscle wasting and cachexia orany combination thereof in an individual wherein the composition(s) isan inhibitor of any members of the STAT protein family, including STAT1,STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), or STAT6, for example.

In embodiments of the invention, there is a composition selected fromthe group consisting ofN-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,and4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide.The composition may be comprised in a pharmaceutical formulation. Thecomposition may be comprised with a carrier. The composition may becomprised with another therapeutic composition, such as a therapeuticcomposition for a condition selected from muscle weakness, musclewasting and cachexia or any combination thereof. The composition may becomprised in a suitable solvent. The composition may be comprised in asolvent and/or polyethylene glycol (PEG). In specific embodiments, thesolvent is Labrasol® (Caprylocaproyl macrogol-8 glycerides EP;Caprylocaproyl polyoxyl-8 glycerides NF; PEG-8 Caprylic/CapricGlycerides (USA FDA IIG), water, ethanol, glycerin, propylene glycol,isopropyl alcohol, methanol, acetone, isopropanol, acetonitrile,t-butanol, n-hexane, cyclohexane, and so forth. In specific embodiments,the PEG is PEG-200, PEG-300, or PEG-400. In particular cases, thecomposition is formulated in 60% Labrasol® and 40% PEG-400. Thecomposition may be comprised in a tablet, soft gel cap, and so forth.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G demonstrates inhibition of Stat3 binding to immobilizedphosphopeptide ligand by compounds. Binding of recombinant Stat3 (500nM) to a BiaCore sensor chip coated with a phosphododecapeptide based onthe amino acid sequence surrounding Y1068 within the EGFR was measuredin real time by SPR (Response Units) in the absence (0 μM) or presenceof increasing concentrations (0.1 to 1,000 μM) of Cpd3 (FIG. 1A), Cpd30(FIG. 1B), Cpd188 (FIG. 1C), Cpd3-2 (FIG. 1D), Cpd3-7 (FIG. 1E) andCpd30-12 (FIG. 1F). Data shown are representative of 2 or moreexperiments. The equilibrium binding levels obtained in the absence orpresence of compounds were normalized (response obtained in the presenceof compound÷the response obtained in the absence of compound×100),plotted against the log concentration (nM) of the compounds (FIG. 1G).The experimental points fit to a competitive binding curve that uses afour-parameter logistic equation (see exemplary methods for details).These curves were used to calculate IC₅₀ (Table 1).

FIGS. 2A-2F demonstrates inhibition of IL-6-mediated activation of Stat3by compounds. HepG2 cells were pretreated with DMSO alone or DMSOcontaining Cpd3 (FIG. 2A), Cpd188 (FIG. 2B), Cpd30 (FIG. 2C), Cpd3-2(FIG. 2D), Cpd3-7 (FIG. 2E) or Cpd30-12 (FIG. 2F) at the indicatedconcentration for 60 min. Cells were then stimulated with IL-6 (30ng/ml) for 30 min. Protein extracts of cells were separated by SDS-PAGE,blotted and developed serially with antibodies to pStat3, total Stat3and β-actin. Blots were stripped between each antibody probing. Thebands intensities of immunoblot were quantified by densitometry. Thevalue of each pStat3 band's intensity was divided by each correspondingvalue of total Stat3 band intensity and the results normalized to theDMSO-treated control value and plotted as a function of the log compoundconcentration. The best-fit curves were generated based on 4 ParameterLogistic Model/Dose Response One Site/XLfit 4.2, IDBS. Each panel isrepresentative of 3 or more experiments.

FIGS. 3A-3F provides exemplary chemical formulas and names of compounds.The chemical formulas and names are indicated for Cpd3 (FIG. 3A), Cpd30(FIG. 3B), Cpd188 (FIG. 3C), Cpd3-2 (FIG. 3D), Cpd3-7 (FIG. 3E) andCpd30-12 (FIG. 3F).

FIG. 4 shows effect of compounds on Stat1 activation. HepG2 cells werepretreated with DMSO alone or DMSO containing each of the compounds at aconcentration of 300 μM for 60 min. Cells were then stimulated withIFN-γ (30 ng/ml) for 30 min. Protein extracts of cells were separated bySDS-PAGE and immunoblotted serially with antibodies to pStat1, totalStat1 and β-actin. Blots were stripped between each immunoblotting. Theresults shown are representative of 2 or more experiments.

FIGS. 5A-5C provides comparisons of the Stat3 and Stat1 SH2 domainsequences, 3-D structures and van der Waals energies of compoundbinding. Sequence alignment of Stat3 and Stat1 SH2 domains is shown inFIG. 5A. The residues that bind the pY residue are highlighted in andpointed to by a solid arrow, the residue (E638) that binds to the +3residue highlighted and pointed to by a dotted arrow and Loop_(βC-βD)and Loop_(αB-αC), which comprise the hydrophobic binding siteconsisting, are highlighted and pointed to by dot-dashed and dashedarrows, respectively. FIG. 5B shows an overlay of a tube-and-fog van derWaals surface model of the Stat3 SH2 domain and a tube-and-fog van derWaals surface model of the Stat1 SH2. The residues of the Stat3 SH2domain represents Loop_(βC-βD) are highlighted and shown by dottedcircles and the residues represent Loop_(αB-αC) are highlighted andshown by a dotted-dashed circle; the corresponding loop residues withinthe Stat1 SH2 domain are shown in a light fog surrounding the circles.This overlay is shown bound by Cpd3-7 as it would bind to the Stat3 SH2domain. The van der Waals energy of each compound bound to the Stat1 SH2domain or the Stat3 SH2 domain was calculated, normalized to the valuefor Stat1 and depicted in FIG. 5C.

FIGS. 6A-6F shows a computer model of each compound bound by the Stat3SH2 domain. The results of computer docking to the Stat3 SH2 domain isshown for Cpd3 (FIG. 6A), Cpd30 (FIG. 6B), Cpd188 (FIG. 6C), Cpd3-2(FIG. 6D), Cpd3-7 (FIG. 6E) and Cpd30-12 (FIG. 6F). The image on theleft of each panel shows the compound binding to a spacefilling model ofthe Stat3 SH2 domain. The pY-residue binding site is represented bydashed circle, the +3 residue binding site is represented by a solidcircle, loop Loop_(βC-βD) is represented by dotted circle and loopLoop_(αB-αC) is represented by dot-dashed circle. Residues R609 and K591critical for binding pY are shown within a dashed circle, residue E638that binds the +3 residue shown within a solid circle and thehydrophobic binding site consisting of Loop_(βC-βD) and Loop_(αB-αC) isshown within a dash-dot and dotted circle, respectively. The image onthe right side of each panel is a closer view of this interaction withhydrogen bonds indicated by dotted lines. In FIG. 6A the negativelycharged benzoic acid moiety of Cpd3 has electrostatic interactions withthe positively-charge pY residue binding site consisting mainly of theguanidinium cation group of R609 and the basic ammonium group of K591.The benzoic acid group also forms a hydrogen-bond network consisting ofdouble H-bonds between the carboxylic oxygen and the ammonium hydrogenof R609 and the amide hydrogen of E612. H-bond formation also occursbetween the benzoic acid carbonyl oxygen and the side chain hydroxylhydrogen of Serine 611. Within the +3 residue-binding site, the oxygenatom of 1,4-benzodioxin forms a hydrogen bond with the amide hydrogen ofE638. In addition, the 2,3-dihydro-1,4-benzodioxin of Cpd3 interactswith the loops forming the hydrophobic binding site. In FIG. 6B thecarboxylic terminus of the benzoic acid moiety of Cpd30, which isnegatively charged under physiological conditions, forms a salt bridgewith the guanidinium group of R609 within the pY residue binding site.Within the +3 residue-binding site, the oxygen of the thiazolidin groupforms a H-bond with the peptide backbone amide hydrogen of E638. Inaddition, the thiazolidin moiety plunges into the hydrophobic bindingsite. In FIG. 6C there is an electrostatic interaction between the(carboxymethyl) thio moiety of Cpd188 carrying a negative charge and thepY-residue binding site consisting of R609 and K591 carrying positivecharge under physiological conditions. There are H-bonds between thehydroxyloxygen of the (carboxymethyl) thio group of Cpd188 and theguanidinium hydrogen of R609, between the hydroxyl-oxygen of the(carboxymethyl) thio group and the backbone amide hydrogen of E612, andbetween the carboxyl-oxygen of the (carboxymethyl) thio group of Cpd188and the hydroxyl-hydrogen of S611. Within the +3 residue-binding site,there is a H-bond between the hydroxyl-oxygen of benzoic acid group ofCpd188 and the amide-hydrogen of E638. In addition, the benzoic acidgroup extends and interacts with the hydrophobic binding site. In FIG.6D the benzoic acid group of Cpd3-2 has significant electrostaticinteractions with the pY-residue binding site pocket, mainly contributedby R609 and K591, and forms two H bonds; the carboxylic oxygen of thebenzoic acid group binds the guanidinium hydrogen of R609, and thecarbonyl oxygen of the benzoic acid group binds to the carbonyl hydrogenof S611. Within the +3 residue-binding site, oxygen within the1,3-dihydro-2H-inden-2-ylidene group forms an H bond to the backboneamide-hydrogen of E638. In addition, the 1,3-dihydro-2H-inden-2-ylidenegroup plunges into the hydrophobic binding site. In FIG. 6E H-bonds areformed between the carbonyl-oxygen of the methyl 4-benzoate moiety ofCpd 3-7 and the side chain guanidinium of R609 and between themethoxy-oxygen and the hydrogen of the ammonium terminus of K591. The(2-methoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen group of Cpd3-7blocks access to the amide hydrogen of E638 within the +3residue-binding site. In addition, this group plunges into thehydrophobic binding site. In FIG. 6F there are electrostaticinteractions between the benzoic acid derivative group of Cpd30-12 andR609 and 591 within the pY-residue binding site. Also, H-bonds areformed between the hydroxyl-oxygen of Cpd30-12 and theguanidinium-hydrogen of R609, between the carboxyl-oxygen of Cpd30-12and the hydroxyl-hydrogen of S611 and between the furyl group ofCpd30-12 and the hydrogen of ammonium of K591. The 1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene groups blocks accessto the +3 residue binding site; however, it extends into the groovebetween the pY-residue binding site and LoopβC-βD, while sparing thehydrophobic binding site.

FIGS. 7A-7B shows inhibition of cytoplasmic-to-nuclear translocation ofStat3 assessed by confocal and high-throughput fluorescence microscopy.In FIG. 7A, MEF/GFP-Stat3 cells grown on coverslips were pretreated withDMSO that either contained (row four) or did not contain (row three)Cpd3 (300 μM) for 60 min before being stimulated without (row one) orwith IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes (rows two,three and four). Coverslips were examined by confocal fluorescentmicroscopy using filters to detect GFP (column one), DAPI (column two)or both (merge; column three). In FIG. 7B, MEF-GFP-Stat3 cells weregrown in 96-well plates with optical glass bottoms and pretreated withthe indicated compound at the indicated concentrations in quadruplicatefor 1 hour then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml)for 30 minutes. Cells were fixed and the plates were examined byhigh-throughput microscopy to determine the fluorescence intensity inthe nucleus (FLIN) and the % ΔFLIN_(Max) was calculated as described inExample 1. Data shown are mean±SD and are representative of 2 or morestudies. Best-fit curves were generated based on 4 Parameter LogisticModel/Dose Response One Site/XLfit 4.2, IDBS and were used to calculateIC₅₀ (Table 1).

FIG. 8 demonstrates inhibition of Stat3 DNA binding by compounds.Electrophoretic mobility shift assays were performed using whole-cellextracts prepared from HepG2 cells without and with stimulation withIL-6 (30 ng/ml) for 30 min. Protein (20 μg) was incubated withradiolabeled duplex oligonucleotide (hSIE) and DMSO without or with theindicated compounds (300 uM) for 60 minutes at 37° C. then separated byPAGE. The gel was dried and autoradiographed; the portion of the gelcorresponding to the Stat3-bound hSIE band is shown. Data shown arerepresentative of 2 studies.

FIG. 9 shows Cpd3, Cpd30 and Cpd188 and the hydrophobicity orhydrophilicity of the surface of the molecule. The dashed arrows pointto hydrophilic surfaces, and the solid arrows point to hydrophobicsurfaces.

FIG. 10 illustrates exemplary compound 3 (Cpd3). The top-left picture ofFIG. 11 shows Cpd3 docked into Stat3 and the interaction between Cpd3and the surface of the protein and derivatives of Cpd3 that can fit intothe surface of the protein. Stars represent atoms and chemical groupsthat can be replaced with other atoms or chemical groups to create oneor more functional derivatives. The hydrophobic/hydrophilic surfaces ofCpd3 are also demonstrated on the top-right picture. The dashed arrowspoint to hydrophilic surfaces, and the solid arrows point to hydrophobicsurfaces. R₁ and R₂ could be identical or different and may comprisehydrogen, carbon, sulfur, nitrogen, oxygen, alkanes. cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, or benzoic acid-basedderivatives.

FIG. 11 illustrates exemplary compound 30 (Cpd30). The top-left pictureof FIG. 12 shows Cpd30 docked into Stat3 and the interaction betweenCpd30 and the surface of the protein, and derivatives of Cpd30 that fitinto the surface of the protein. Stars represent atoms and chemicalgroups that can be replaced with other atoms or chemical groups tocreate one or more functional derivatives. The hydrophobic/hydrophilicsurfaces of Cpd30 are also demonstrated on the top-right picture. Thedashed arrows point to hydrophilic surfaces, and the solid arrows pointto hydrophobic surfaces. 2-D structure of Cpd30 shown on the bottompicture, R₁, R₂ R₃ and R₄ could identical or different and may comprisebe hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes. cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, aor benzoic acid-basedderivatives.

FIG. 12 illustrates exemplary compound 188 (Cpd188). The top picture ofFIG. 12 shows Cpd188 docked into Stat3 SH2 domain and the interactionbetween Cpd188 and the surface of the protein, and derivatives of Cpd188that fit into the surface of the protein. Stars represent atoms andchemical groups that can be replaced with other atoms or chemical groupsto create one ore more functional derivative. Thehydrophobic/hydrophilic surfaces of Cpd188 are also demonstrated on theleft picture on the bottom. The dashed arrows point to hydrophilicsurfaces, and the solid arrows point to hydrophobic surfaces. Shown onthe right bottom picture, R₁ and R₂ could be identical or different andmay comprise hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes. cyclicalkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, or benzoic acid-basedderivatives.

FIG. 13 illustrates schematic diagrams of Stat1 and Stat3.

FIG. 14 demonstrates that SPR IC₅₀ of 2nd generation Stat3 chemicalprobes is inversely correlated with 3-D pharmacophore score.

FIG. 15 shows SPR IC₅₀ and AML apoptosis EC₅₀ of parent Cpd188 and two2nd generation 188-like Stat3 chemical probes.

FIG. 16 provides an illustration of structure-activity relationships of38 Cpd188-like, 2nd generation Stat3 probes.

FIG. 17 shows an exemplary modification scheme for 3rd generation Stat3probe development using Cpd188-15 as a scaffold.

FIG. 18 provides illustration of the electrostatic surface of Stat3 SH2domain (positive area in blue, neutral in white and negative in red in acolor figure) and 20 docking poses of 5 (R═CH₂PO₃ 2-), showing stronginteractions between phosphonate groups (in purple and red) andK591/R609.

FIGS. 19A-19C shows inflammatory cytokines and p-Stat3 are elevated inmuscles of patients with CKD. FIG. 19A. Immunostaining of musclesections for IL-6 and TNFα (brown color) from biopsies of age- andgender-matched, healthy control subjects (left panel) and CKD patients(middle panel). Staining quantification is calculated as the percentageof muscle fibers that are immunostained (right panel; n=3 controlsubjects; n=4 CKD patients; ruler=50 μm). FIG. 19B. Representativewestern blots for p-Stat3 in control subjects and CKD patients (upperpanel) and the ratio of the intensity of p-Stat3 to total Stat3 (lowerpanel) (n=6 control subjects; n=6 CKD patients). FIG. 19C. Musclesections from control subjects and CKD patients were immunostained forp-Stat3 (upper panel). Brown nuclei are p-Stat3 positive (arrows).Percentage of p-Stat3 positive nuclei in a total of 550 nuclei (lowerpanel; n=4 control subjects; n=6 CKD patients). Values are means±SEM.*p<0.05 vs. control subjects.

FIGS. 20A-20J shows muscle-specific Stat3 knockout in mice suppressesCKD or streptozotocin-induced muscle wasting. FIG. 20A. Density ofp-Stat3 corrected for total Stat3 in lysates of gastrocnemius muscles(upper panel; n=5 mice/group; *p<0.05 vs. sham control mice). Also shownare representative western blots of p-Stat3 (lower panel). FIG. 20B.Changes in body weights of Stat3 KO and Stat3flox/flox, control miceover 5 weeks following creation of CKD (n=10 pairs of mice; *p<0.05 vs.Stat3flox/flox). FIGS. 20C & 20D. Average weights of mixed fibergastrocnemius and tibialis anterior (TA) muscles (n=10 mice/group).FIGS. 20E & 20F. EDL muscles from sham or CKD mice and eitherStat3flox/flox or Stat3 KO were isolated. Rates of protein synthesis(FIG. 20E) and protein degradation (FIG. 20F) were measured (n=20 EDLmuscles from 10 mice/group). FIG. 20G. Muscle force of each mouse usedin FIG. 2B was measured on four consecutive days. The average muscleforce (in Newtons) is shown (n=10 mice/group). FIG. 20H. Representativewestern blots of p-Stat3 in lysates of gastrocnemius muscles of acutelydiabetic (STZ) and control mice. Bar graph shows the densities ofp-Stat3 corrected for GAPDH (n=10 mice/group; *p<0.05 vs. CTRL mice).FIGS. 20I & 20J. Average weights of the mixed fiber tibialis anterior(TA) and gastrocnemius muscles from both legs (n=10 mice/group; *p<0.05vs. control Stat3^(flox/flox)). Values are means±SEM.

FIGS. 21A-21G provides a small molecule inhibitor of Stat3 activation,C188-9, that blocks CKD-induced muscle wasting. FIG. 21A. Sham or CKDmice were treated with C188-9 or D5W (diluent) for 14 days.Representative western blots of p-Stat3, Stat3 and GAPDH from lysates ofgastrocnemius muscles are shown (n=8 mice/group). FIG. 21B. Differencesin body weights of pair-fed, sham or CKD mice treated with C188-9 or D5Wat baseline and after 7 or 14 days of treatment (*p<0.05 vs. D5W sham).FIGS. 21C & 21D. Average weights of mixed fiber gastrocnemius andtibialis anterior (TA) muscles from both legs (n=7 mice/group). FIG.21E. Cryosections of TA muscles were immunostained with anti-laminin toidentify the muscle basement membrane. The myofiber areas were measuredand the myofiber size distribution was calculated from the areas of ˜500myofibers assessed by an observer blinded to treatment group (n=4 pairsof mice). FIG. 21F. Muscle force of each mouse studied in FIG. 3C wasmeasured on four consecutive days (Experimental Procedures; n=7mice/group). G&H. At 2 weeks of C188-9 or D5W treatment, proteinsynthesis (FIG. 21G) and degradation (FIG. 21H) were measured (n=8 pairsof mice; *p<0.05 vs. D5W). Values are means±SEM.

FIGS. 22A-22G demonstrates that Stat3 activation in C2Cl2 myotubesincreases the expression of C/EBPδ and myostatin. FIG. 22A.Representative western blots from C2Cl2 myotubes treated with IL-6 (100ng/ml) for different times (left panel). Fold-changes in the densitiesof proteins corrected for GAPDH at different times calculated fromvalues at time zero (right panel), n=3 repeats; *p<0.05 vs. time zero.FIG. 22B. C2Cl2 myotubes were infected with a lentivirus expressingconstitutively active Stat3 (Stat3C-GFP). A representative western blotfor the indicated proteins is shown. FIG. 22C. C2Cl2 myotubes weretreated with or without C188-9 for 2 h before adding IL-6 (100 ng/ml)for 24 h. A representative western blot for the indicated proteins isshown. FIG. 22D. C2Cl2 myoblasts were co-transfected with a plasmidexpressing C/EBPδ promoter-driven luciferase, Renila plus a lentivirusexpressing Stat3C-GFP and treated with or without IL-6. Dual luciferaseactivity was measured (n=3 repeats; *p<0.05 vs respective GFP control).FIG. 22E. C2Cl2 myoblasts were transfected with control siRNA or C/EBPδsiRNA and after differentiation to myotubes were treated with or withoutIL-6. Representative western blots of Stat3, C/EBPδ and myostatin areshown. FIG. 22F. C2Cl2 myoblasts were co-transfected with a plasmidexpressing the myostatin promoter-driven luciferase plus plasmids (cDNA3control, Stat3C, C/EBPδ, C/EBPδ siRNA or Stat3C plus C/EBPδ siRNA) andtreated with or without IL6. Luciferase activity was measured (n=3repeats; *p<0.05 vs. cDNA3 CTRL). FIG. 22G. C2Cl2 myoblasts weretransfected with lentivirus expressing a siRNA to myostatin. Myoblastsexhibiting suppression of myostatin were selected and thendifferentiated after they had been transfected with plasmids expressingStat3C, C/EBPδ or Stat3C plus C/EBPδ. In these cells, proteindegradation (upper panel; n=6 repeats, #p<0.05 vs GFP control, *p<0.05vs siRNA CTRL) was measured. Western blots of proteins expressed inresponse to tranfections was shown in FIG. 32.

FIGS. 23A-23G demonstrates that Stat3 activation in mouse musclesincreases C/EBPδ and myostatin expression. FIG. 23A. Representativewestern blots of the indicated proteins from lysates of gastrocnemiusmuscles of control (Stat3flox/flox) or Stat3 KO sham or CKD mice. FIGS.23B & 23C. mRNAs of myostatin (FIG. 23B) and C/EBPδ in muscles of shamor CKD mice analyzed by RT-PCR (n=4 mice/group; *p<0.05 vs.Stat3flox/flox sham). FIG. 23D. Representative western blots of theindicated proteins in lysates of gastrocnemius muscles of STZ vs. WTcontrol mice (n=5 pairs). FIG. 23E. Sham or CKD mice were treated withC188-9 or D5W (diluent) for 14 days. Representative western blots ofindicated proteins from lysates of gastrocnemius muscles are shown (n=8mice/group). FIGS. 23F & 23G. mRNA levels of myostatin (FIG. 23F) andC/EBPδ (FIG. 23G) analyzed by RT-PCR and corrected for GAPDH (n=3mice/group: wild-type mice without CKD; sham mice treated with C188-9 orD5W; mice with CKD treated with C188-9 or D5W; *p<0.05 vs. WT non-CKD).Values are means±SEM. FIG. 6. C/EBPδ and myostatin mediate CKD orStat3-induced muscle wasting. A. Body weights of wild type or homo- orhetero-C/EBPδ KO mice following creation of CKD. Values are expressed asa percentage of basal body weight (mean±SEM; n=9 for WT mice; n=11 forC/EBPδ−/−; n=11 for C/EBPδ+/− mice; *p<0.05 vs. WT CKD). FIG. 23B.Survival was calculated as the percentage of mice surviving at 3 weeksafter CKD or after sham surgery (n=20 for WT; n=25 for C/EBPδ−/−; n=21for C/EBPδ+/−; *p<0.05 vs. C/EBPδ−/− CKD). FIG. 23C. Average weightsfrom both legs of red fiber (soleus) or white fiber (EDL) muscles(mean±SEM; n=10 mice/group; *p<0.05 vs. WT CKD). FIG. 23D.Representative western blots of p-Stat3 and myostatin from muscles ofCKD or sham-operated mice of the following groups: C/EBPδ−/−, C/EBPδ+/−or control (WT). FIG. 23E. Cryosections of gastrocnemius muscles frommice that were transfected with lentivirus expressing Stat3C-GPF or GFPand treated with anti-myostatin inhibitor or PBS. The sections wereimmunostained with p-Smad2/3 (red, lower panel). The upper panel,overlap picture shows GFP-positive myofibers (green) that expressedp-Smad2/3. FIG. 23F. GFP-positive areas in myofibers were measured andthe mean myofiber sizes of each group are shown (left panel). Thepercentage of p-Smad2/3 positive nuclei to total nuclei was calculated(right panel; mean±SEM, *p<0.05 vs. GFP/PBS).

FIGS. 24A-24F demonstrates a link from Stat3 to C/EBPδ to myostatin invivo. FIG. 24A. Body weight as a function of time for pair fed,homozygous C/EBPδ KO mice with CKD compared to control. FIG. 24B.Survival percentages over time in C/EBPδ KO mice with CKD compared tovarious controls. FIG. 24C. Muscle weight in C/EBPδ KO mice with CKDcompared to wild type. FIG. 24D. Expression levels of myostatin in micewith homozygous C/EBPδ KO. FIG. 24E demonstrates immunostaining withanti-MSTN peptibody in mice expressing constitutively active Stat3-GFP(Stat3C-GFP), and FIG. 24F. Quantification of GFP in a myofiber area.

FIGS. 25A-25D shows evidence for a p-Stat3, C/EBPδ and myostatin pathwayin muscles of patients with CKD FIG. 25A. Representative western blotsof p-Akt from muscle biopsies of healthy control or CKD patients. Bargraph shows the densities of p-Akt corrected for GAPDH (lower panel; n=4CKD patients and 3 healthy subjects). FIG. 25B. Levels of mRNAs ofC/EBPδ or myostatin were analyzed by RT-PCR from muscle biopsies ofhealthy control or CKD patients (n=5 control subjects and 9 CKDpatients). FIG. 25C. Representative western blots of the indicatedproteins from muscle biopsies from healthy control or CKD patients. FIG.25D. The band densities were quantified after correction for GAPDH (n=3pairs for CEBPδ; n=8 pairs for myostatin). Values are means±SEM. *p<0.05vs. healthy controls.

FIG. 26: Muscles of patients with CKD exhibited increased mRNA levels ofTNFα. RT-PCR was used to assess TNFα mRNA levels corrected for GAPDH.The bar graph (mean±SEM) illustrates the difference found in samples of9 patients with CKD and 5 healthy control (*, p<0.05 vs. healthysubjects).

FIG. 27 shows the body weight changes in mice with Stat3 KO in muscle.Changes in body weights of Stat3 KO and Stat3flox/flox control micemeasured from 3 to 8 weeks after birth. There were no significantdifferences in body weights of Stat3 KO vs. Stat3flox/flox mice withoutCKD (n=6 control; n=9 Stat3 KO).

FIG. 28 provides serum levels of IL-6 from STZ mice was assessed byELISA (n=4 mice/group).

FIG. 29 shows changes in body weights of Stat3 KO or Stat3flox/flox micewith acute diabetes. During 9 days after streptozotocin injection, thedaily body weight changes was shown (n=10 mice/group; *p<0.05 vs.Stat3flox/flox non-STZ; #p<0.05 vs. Stat3 KO STZ).

FIG. 30 demonstrates Stat3 activation in C2Cl2 myotubes increases themRNA expression of C/EBPδ C2Cl2 myotubes were treated with IL-6 (100ng/ml) for different times. RT-PCR was used to assess mRNA levels ofC/EBPδ. Bar graphs show changes in mRNAs of C/EBP after correction forthe mRNA of GAPDH. n=3 repeats; *p<0.05 vs. time zero.

FIG. 31 provides Stat3 activation in C2Cl2 myotubes increases the mRNAexpression of myostatin. C2Cl2 myotubes were treated with IL-6 (100ng/ml) for different times. RT-PCR was used to assess mRNA levels ofmyostatin. Bar graphs show changes in mRNAs of myostatin aftercorrection for the mRNA of GAPDH. n=3 repeats; *p<0.05 vs. time zero.

FIG. 32 demonstrates the mRNA expression of myostatin and C/EBPδ inducedby IL-6 requires Stat3 activation. C2Cl2 myotubes were treated with orwithout C188-9 for 1 or 24 h and RT-PCR was used to assess mRNA levels.The bar graphs (mean±SEM) illustrate the mRNA levels corrected for GAPDHof myostatin (top), and C/EBPδ (bottom). N=3 independent experiments; *,p<0.05 vs. results without C188-9.

FIG. 33 demonstrates that C-188-9 suppresses IL-6 induced myotubewasting. C2Cl2 myotubes were treated with C188-9, a Stat3 inhibitor, for2 h before adding IL-6 (100 ng/ml) for 24 h. Myotube sizes were measured(mean±SEM; n=3 independent experiments; *, p<0.05 vs. untreatedmyotubes).

FIG. 34 shows protein levels were measured in C2Cl2 cells with myostatinknockdown and overexpress Stat3C or C/EBPδ. C2Cl2 myoblasts weretransfected with lentivirus expressing a siRNA to myostatin. Myoblastsexhibiting suppression of myostatin were selected and thendifferentiated after they had been transfected with plasmids expressingStat3C, C/EBPδ or Stat3C plus C/EBPδ. Western blots of proteinsexpressed in response to tranfections were shown.

FIG. 35 shows that myostatin inhibition blocked Stat3C inducedp-Smad2/3. The muscle lysates from muscle treafected with lentivirusexpressing GFP or Stat3C and mice treated with anitmyostatin peptibodyor PBS were subjected to western bloting to show the levels of p-stat3and p-Smad2/3.

FIGS. 36A-36D shows that conditioned media from C26 or LLC-cancer cellsactivates p-Stat3 in C2Cl2 cells, a model of skeletal muscle. C2Cl2myotubes. FIG. 36A. Representative western blots of p-Stat3 and Stat3 inC2Cl2 myotubes that were exposed for different times to conditionedmedia from C26 colon cancer cells. FIG. 36B. C2Cl2 myotubes werepretreated with the Stat3 inhibitor, C188-9, for 2 hours before theywere exposed to conditioned media from C26 or LLC cancer cells.Representative western blots for p-Stat3 or Stat3 are shown. FIG. 36C.C2Cl2 myotubes were treated with C188-9 plus conditioned media from C26cells for different times. Representative western blots for C/EBPδ andmyostatin are shown. FIG. 36D. The average sizes of C2Cl2 myotubes wasassessed following incubation with conditioned media of C26 cells withor without C188-9 for 72 hours (mean±SEM; p<0.05).

FIGS. 37A-37E shows that muscle-specific Stat3 KO in mice suppressesLLC-induced loss of muscle mass. Mice with muscle-specific Stat3 KO orcontrol mice, Stat3^(flox/flox), (10 mice in each group) were injectedwith LLC 18 days earlier. FIG. 37A. Changes in body weight are expressedas a percentage of the body weight measured before LLC was injected.FIG. 37B. Weights of tumor measured when mice were sacrificed. FIG. 37C.Weights of different muscles (TA, tibialis anterior; Gast,gastrocnemius; and EDL, extensor digitorum longus) measured at 18 daysafter injecting LLC. FIG. 37D. Representative western blotting of Stat3,C/EBPδ and myostatin from muscles of mice with muscle-specific KO ofStat3 or Stat3^(flox/flox) mice. Mice with and without tumor werecompared and densities of blots were quantified (lower panel). FIG. 37E.Muscle grip strength (n=5 mice in each group) was measured (mean±SEM).

FIGS. 38A-38E demonstrates that elimination of C/EBPδ in mice suppressesLLC-induced cachexia. C/EBPδ KO and control mice were injected with LLCand 18 days later, there was measured: FIG. 38A. body weight; FIG. 38B.weights of different types of muscle based on fiber type; FIG. 38C.measured rates of muscle protein degradation; FIG. 38D. muscle gripstrength; FIG. 38E. representative western blots of myostatin in musclesof m C/EBPδ or control mice treated with or without LLC (upper panel).The fold-increase in myostatin vs. results in control mice are shown inthe lower panel. Results are reported as mean±SEM.

FIGS. 39A-39G provides that blocking Stat3 activation with C188-9, aStat3 inhibitor, suppresses cancer cachexia. CD2F1 mice bearing C26tumor for 5 days were treated with C188-9 twice daily for 14 days.Results from these mice were compared to those of CD2F1 mice bearingtumor and treated with the diluent, 5% dextose in water (D5W). CD2F1mice without C26 tumors served as the control mice. There were 12 micein each group. FIG. 39A. representative western blots of differentproteins (upper panel) were quantified (lower panel). Results shown are:FIG. 39B. body weights; FIG. 39C. muscle weights; FIG. 39D. thedistribution of myofiber sizes of in the 3 groups of mice; FIG. 39E.muscle grip strength; and FIGS. 39F & 39G are measured rates of proteinsynthesis and degradation. Results are mean±SEM.

FIGS. 40A-40E shows that p-Stat3 stimulates the transcription ofcaspase-3, participating in the development of cancer cachexia. FIG.40A. representative western blot reveals increased levels ofprocaspase-3 and caspase-3 in muscles of mice bearing C26 or LLC. FIG.40B. representative western blot demonstrating increased caspase-3activity measured as the cleavage of actin to produce the 14 kDa actinfragment, characteristic of catabolic conditions. FIG. 40C. C2Cl2myotubes were treated for 24 hours with conditioned media from C26cells. A ChiP assay shows that p-Stat3 binds to the caspase-3 promoter.FIG. 40D. C2Cl2 myotubes were infected with an adenovirus expressing GFPor Stat3. After 24 hours, cells expressing Stat3C were stimulated byIL-6. Results of a ChiP assay using anti-p-Stat3 or anti-Stat-3 revealedbinding of Stat3 to the caspase-3 promoter. FIG. 40E. C2Cl2 cells weretransfected with different segments of a caspase-3 promoter-luciferaseconstruct plus a plasmid that expresses constitutively active Stat3.Subsequently, cells were treated with or without IL-6 for 6 h. andluciferase activity was used to assess caspase-3 promoter activity.Results are mean±SEM.

FIGS. 41A-41F shows activation of Stat3 induces ubiquitin-proteasomesystem in cancer-induced cachexia. FIG. 41A. C2Cl2 myotubes were treatedwith conditioned media from C26 cells with or without C188-9 for 72hours. A representative western blot showing a decrease in the myosinheavy chain is blocked by C188-9. FIG. 41B. C2Cl2 myotubes were treatedwith conditioned media from C26 cells with or without C188-9 for 24hours. Levels of mRNAs of MAFbx/Atrogin-1 and MuRF-1 are shown. FIGS.41C & 41D. CD2F1 mice bearing C26 tumors were treated with C188-9 for 2weeks. Representative western blots from lysates of gastrocnemiusmuscles show that C188-9 suppresses MAFbx/Atrogin-1 protein and mRNAs inmice bearing C26 tumors. FIG. 41E. LLC tumors in mice withmuscle-specific KO of Stat3 or Stat3^(flox/flox) and after 14 days, arepresentative western blot from muscle shows the protein level ofMAFbx/Atrogin-1. Results are mean±SEM.

FIG. 42 illustrates a summary figure showing how cancer that activatesp-Stat3 in muscle can stimulate loss of muscle mass. Stat3 activationstimulates expression of C/EBPδ which increases myostatin andMAFbx/Atrogin-1 and MuRF-1 to increase muscle wasting by the UPS. Stat3activation also increases caspase-3 expression and activity tocoordinate muscle proteolysis with the UPS.

DETAILED DESCRIPTION OF THE INVENTION

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

In some embodiments, there is a method of treating, preventing, and/orreducing the risk of a condition selected from muscle weakness, musclewasting and cachexia or any combination thereof in an individual,comprising delivering to the individual one or more particularcompounds. In some embodiments, the compound(s) is a STAT3 inhibitor. Incertain embodiments the compound(s) is not a STAT3 inhibitor. Inparticular cases, the compound(s) is a STAT1 inhibitor, but inparticular cases it is not a STAT1 inhibitor. In certain aspects, thereare some compounds that are both STAT3 and STAT1 inhibitors or isneither a STAT3 or STAT1 inhibitor.

In certain embodiments of the invention, there is a compound for use inthe prevention, treatment, and/or reduction in risk for a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof, wherein the compound is selected from the groupconsisting ofN-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide,or a combination thereof, a functionally active derivative, and amixture thereof.

In certain embodiments of the invention, there is a compound for use inthe prevention, treatment, and/or reduction in risk for a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof, wherein the compound is selected from the groupconsisting of4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoicacid;4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoicacid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid;3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid; methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate;4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoicacid; a functionally active derivative and a mixture thereof. In aspecific embodiment of the invention, the composition is a Stat3inhibitor but does not inhibit Stat1.

In a specific embodiment of the invention, the composition is deliveredin vivo in a mammal. In another embodiment the mammal is a human. Inanother specific embodiment the human is known to have a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof, is suspected of having a condition selected frommuscle weakness, muscle wasting and cachexia or any combination thereof,or is at risk for developing a condition selected from muscle weakness,muscle wasting and cachexia or any combination thereof. In anotherembodiment, the human is known to have a condition selected from muscleweakness, muscle wasting and cachexia or any combination thereof and isreceiving an additional therapy for the a condition selected from muscleweakness, muscle wasting and cachexia or any combination thereof and/oran underlying condition that is related to a condition selected frommuscle weakness, muscle wasting and cachexia or any combination thereof.Composition(s) of the disclosure treat, prevent, and/or reduce the riskof body weight loss and/or muscle weight loss, in particularembodiments.

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. Still further,the terms “having”, “including”, “containing” and “comprising” areinterchangeable and one of skill in the art is cognizant that theseterms are open ended terms. Some embodiments of the invention mayconsist of or consist essentially of one or more elements, method steps,and/or methods of the invention. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

The term “inhibitor” as used herein refers to one or more molecules thatinterfere at least in part with the activity of Stat3 to perform one ormore activities, including the ability of Stat3 to bind to a moleculeand/or the ability to be phosphorylated.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention that is effective for producing some desiredtherapeutic effect, e.g., treating (i.e., preventing and/orameliorating) cancer in a subject, or inhibiting protein-proteininteractions mediated by an SH2 domain in a subject, at a reasonablebenefit/risk ratio applicable to any medical treatment. In oneembodiment, the therapeutically effective amount is enough to reduce oreliminate at least one symptom. One of skill in the art recognizes thatan amount may be considered therapeutically effective even if the canceris not totally eradicated but improved partially. For example, thespread of the cancer may be halted or reduced, a side effect from thecancer may be partially reduced or completed eliminated, life span ofthe subject may be increased, the subject may experience less pain, andso forth.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “at risk for having muscle wasting” as used herein refers toan individual that is at risk for having less than their normal level ofstrength or too little muscle or having loss in muscle, such as anindividual that has an underlying medical condition with such a symptomor is elderly.

The phrase “at risk for having cachexia” is used herein to refer toindividuals that have a chance to have cachexia because of past,present, or future factors. In particular embodiments, an individual atrisk for having cachexia is one that has an underlying condition that isknown to cause or be associated with cachexia as at least one symptom.The condition may or may not be chronic. In some embodiments, anunderlying medical condition that is known to have cachexia as at leastone symptom includes at least renal failure, cancer, AIDS, HIVinfection, chronic obstructive lung disease (including emphysema),multiple sclerosis, congestive heart failure, tuberculosis, familialamyloid polyneuropahty, acrodynia, hormonal deficiency, metaoblicacidosis, infectious disease, chronic pancreatitis, autoimmune disorder,celiac disease, Crohn's disease, electrolyte imbalance, Addison'sdisease, sepsis, burns, trauma, fever, long bone fracture,hyperthyroidism, prolonged steroid therapy, surgery, bone marrowtransplant, atypical pneumonia, brucellosis, endocarditis, Hepatitis B,lung abscess, mastocytosis, paraneoplastic syndrome, polyarteritisnodosa, sarcoidosis, systemic lupus erythematosus, myositis,polymyositis, dematomyosytis, rheumatological diseases, autoimmunedisease, collogen-vascular disease, visceral leishmaniasis, prolongedbed rest, and/or addiction to drugs, such as amphetamine, opiates, orbarbitutates.

As used herein, “binding affinity” refers to the strength of aninteraction between two entities, such as a protein-protein interaction.Binding affinity is sometimes referred to as the K_(a), or associationconstant, which describes the likelihood of the two separate entities tobe in the bound state. Generally, the association constant is determinedby a variety of methods in which two separate entities are mixedtogether, the unbound portion is separated from the bound portion, andconcentrations of unbound and bound are measured. One of skill in theart realizes that there are a variety of methods for measuringassociation constants. For example, the unbound and bound portions maybe separated from one another through adsorption, precipitation, gelfiltration, dialysis, or centrifugation, for example. The measurement ofthe concentrations of bound and unbound portions may be accomplished,for example, by measuring radioactivity or fluorescence, for example.K_(a) also can be inferred indirectly through determination of the K_(i)or inhibitory constant. Determination of the K_(i) can be made severalways for example by measuring the K_(a) of STAT3 binding to itsphosphopeptide ligand within the EGFR at position Y1068 and by measuringthe concentration of a molecule that reduces binding of STAT3 by 50%. Incertain embodiments of the invention, the binding affinity of a Stat3inhibitor for the SH2 domain of Stat3 is similar to or greater than theaffinity of the compounds listed herein.

The term “domain” as used herein refers to a subsection of a polypeptidethat possesses a unique structural and/or functional characteristic;typically, this characteristic is similar across diverse polypeptides.The subsection typically comprises contiguous amino acids, although itmay also comprise amino acids that act in concert or that are in closeproximity due to folding or other configurations. An example of aprotein domain is the Src homology 2 (SH2) domain of Stat3. The term“SH2 domain” is art-recognized, and, as used herein, refers to a proteindomain involved in protein-protein interactions, such as a domain withinthe Src tyrosine kinase that regulates kinase activity. The inventioncontemplates modulation of activity, such as activity dependent uponprotein-protein interactions, mediated by SH2 domains of proteins (e.g.,tyrosine kinases such as Src) or proteins involved with transmission ofa tyrosine kinase signal in organisms including mammals, such as humans.

As used herein, a “mammal” is an appropriate subject for the method ofthe present invention. A mammal may be any member of the highervertebrate class Mammalia, including humans; characterized by livebirth, body hair, and mammary glands in the female that secrete milk forfeeding the young. Additionally, mammals are characterized by theirability to maintain a constant body temperature despite changingclimatic conditions. Examples of mammals are humans, cats, dogs, cows,mice, rats, and chimpanzees. Mammals may be referred to as “patients” or“subjects” or “individuals”.

II. General Embodiments

General embodiments include one or more compositions for the treatmentand/or prevention and/or reduction in risk or severity of a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof and methods of use. The muscle weakness and/ormuscle wasting and/or cachexia may have an unknown cause or it may beassociated with an underlying condition. The underlying condition may bea catabolic condition. The underlying condition may be chronic kidneydisease, diabetes, cancer, AIDS, and so forth.

In some cases an individual is suspected of having a condition selectedfrom muscle weakness, muscle wasting and cachexia or any combinationthereof; such suspicion may be because the individual has unintentionalmuscle and/or weight loss. In certain aspects, such suspicion may bebecause the individual has muscle loss. In some cases, an individual mayhave at least one symptom of a condition selected from muscle weakness,muscle wasting and cachexia or any combination thereof but may haveother symptoms as well.

In certain cases, an individual is at risk of having a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof. In such cases, the individual has a medicalcondition that can be associated with muscle wasting and/or muscleweakness and/or cachexia and has not had enough progression of themedical condition to manifest muscle wasting and/or muscle weaknessand/or cachexia or has not yet had a detectable symptom of musclewasting and/or muscle weakness and/or cachexia.

In some embodiments, the individual is known to have an underlyingcondition that often has muscle wasting and/or muscle weakness and/orcachexia as at least one symptom, and that individual may or may havenot shown a sign of having muscle wasting and/or muscle weakness and/orcachexia. In cases wherein an individual has an underlying conditionthat often has muscle wasting and/or muscle weakness and/or cachexia asat least one symptom, the individual may be provided with an effectiveamount of one or more compositions of the invention prior to and/orafter the appearance of muscle wasting and/or muscle weakness and/orcachexia. When the individual is provided one of more compositions priorto the appearance of muscle wasting and/or muscle weakness and/orcachexia, the onset of muscle wasting and/or muscle weakness and/orcachexia may be delayed or completely inhibited and/or the severity ofthe muscle wasting and/or muscle weakness and/or cachexia may bereduced, compared to the condition of the individual without havingreceived the composition(s), for example.

In particular embodiments, an individual has been diagnosed with anunderlying condition known to have muscle wasting and/or muscle weaknessand/or cachexia as at least one symptom, and methods of the inventionmay include steps of diagnosing of the muscle weakness and/or musclewasting and/or cachexia and/or the underlying condition of theindividual. An individual may be tested for muscle wasting by standardmeans in the art.

III. Muscle Wasting and/or Muscle Weakness and/or Cachexia

Embodiments of the invention concern methods of treatment and/orprevention of any kind of a condition selected from muscle weakness,muscle wasting, cachexia, and any combination thereof.

Muscle wasting and/or muscle weakness embodiments may arise in thecontext of the individual also having cachexia, or the individual maynot also have cachexia. The muscle wasting and/or muscle weakness may bethe result of age or it may be the result of an underlying medicalcondition. The muscle wasting and/or muscle weakness may manifest priorto or after the detection of other symptoms of the underlying medicalcondition. The muscle wasting may be completely prevented or reversed orthere may be a delay in onset and/or severity upon use of one or morecompositions of the invention.

Muscle wasting and/or muscle weakness may be tested for by a variety ofways, including physical examination; sitting and standing tests;walking tests; measurement of body mass index; reflex tests; blood testsfor muscle enzymes; CT scan; measurement of total body nitrogen; musclebiopsy; and/or electromyogram, for example.

Cachexia, which may also be referred to as wasting syndrome, occurs whenthere is a loss of body mass that cannot be reversed by nutritionalmeans. Examples of symptoms of cachexia include weight loss, muscleatrophy, fatigue, weakness, and/or considerable appetite loss in anindividual that is not actively seeking to lose weight. In particularaspects, the cachexia is the result of a primary pathology, such asgiven that even if the affected individual consumes more calories, thereis loss of body mass. In specific cases, skeletal muscle depletion is aprognostic factor.

In embodiments of the invention, the individual may be known to have themedical condition associated with the muscle wasting and/or muscleweakness and/or cachexia, although in some cases the individual is notknown to have the medical condition. In particular cases, an individualhas muscle wasting and/or muscle weakness and/or cachexia as a symptomof an underlying medical condition that is either known or not known. Anindividual may present with muscle wasting, muscle weakness and/orcachexia as the first symptom and the doctor may then look for anunderlying condition. An individual may present with the underlyingmedical condition and the doctor may monitor the individual for theonset of muscle wasting and/or muscle weakness and/or cachexia or mayrecognize one or more symptoms of muscle wasting and/or muscle weaknessand/or cachexia.

In embodiments of the invention, one or more of the compositions areprovided to an individual with a condition selected from muscleweakness, muscle wasting and cachexia or any combination thereof inaddition to another agent for muscle wasting and/or muscle weaknessand/or cachexia treatment. Examples of cachexia treatment includeanabolic steroids; drugs that mimic progesterone; BMS-945429 (also knownas ALD518); Enobosarm; propranolol and etodolac; omega-3 fatty acids;medical marijuana, IGF-1; nutritional supplements and/or exercise.

In particular embodiments, the individual has cancer cachexia.Approximately half of all cancer patients have cachexia. Althoughcachexia can occur in any type of cancer, those individuals with uppergastrointestinal and pancreatic cancers have the highest frequency ofdeveloping a cachexic symptom. The individual may have terminal cancer.The individual may have metastatic cancer.

In some embodiments the individual has a severe case of cachexia, suchas where the affected individual is so physically weak that theindividual is in a state of immobility resulting from loss of appetite,asthenia, and/or anemia, for example.

IV. Compositions

Embodiments of the invention encompass compositions that are useful fortreating, preventing, and/or reducing the risk of a condition selectedfrom muscle weakness, muscle wasting and cachexia or any combinationthereof. Specific compositions are disclosed herein, but one of skill inthe art recognizes that functional derivatives of such compositions arealso encompassed by the invention. The term “derivative” as used hereinis a compound that is formed from a similar compound or a compound thatcan be considered to arise from another compound, if one atom isreplaced with another atom or group of atoms. Derivative can also referto compounds that at least theoretically can be formed from theprecursor compound.

In particular embodiments, compositions and functionally activederivatives as described herein are utilized in treatment and/orprevention and/or reduction in the risk and/or severity of a conditionselected from muscle weakness, muscle wasting and cachexia or anycombination thereof. Specific but nonlimiting examples of different Rgroups for the compositions are provided in Tables 1, 2, and 3.

In particular embodiments, there are compositions selected from thegroup consisting ofN-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,and functional derivatives thereof.

The term “functionally active derivative” or “functional derivative” isa derivative as previously defined that retains the function of thecompound from which it is derived. In one embodiment of the invention, aderivative ofN-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide,4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoicacid,4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoicacid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid,3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid, methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate,or4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoicacid retains Stat3 inhibitory activity. In another embodiment of theinvention, a derivative of4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoicacid,4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoicacid,4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoicacid,3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid, methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate,or4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoicacid retains Stat3 inhibitory activity and, in specific embodiments,also retains non-inhibition of Stat1, although in some cases it may alsoinhibit Stat1.

In a specific embodiment of the invention, there is a method of treatingand/or preventing and/or reducing the risk and/or severity of acondition selected from muscle weakness, muscle wasting and cachexia orany combination thereof in an individual comprising delivering to theindividual a compound selected from the group consisting ofN-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide,4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoicacid4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoicacid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid,3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid, methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate,4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoicacid, and a mixture thereof.

In another embodiment, the composition comprises the general formula:

wherein R₁ and R₂ may be the same or different and are selected from thegroup consisting of hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes.cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes,alkene-based derivatives, alkynes, alkyne-based derivative, ketones,ketone-based derivatives, aldehydes, aldehyde-based derivatives,carboxylic acids, carboxylic acid-based derivatives, ethers, ether-basedderivatives, esters and ester-based derivatives, amines, amino-basedderivatives, amides, amide-based derivatives, monocyclic or polycyclicarene, heteroarenes. arene-based derivatives, heteroarene-basedderivatives, phenols, phenol-based derivatives, benzoic acid, andbenzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises thegeneral formula:

wherein R₁, and R₃ may be the same or different and are selected fromthe group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen,alkanes. cyclic alkanes, alkane-based derivatives, alkenes, cyclicalkenes, alkene-based derivatives, alkynes, alkyne-based derivative,ketones, ketone-based derivatives, aldehydes, aldehyde-basedderivatives, carboxylic acids, carboxylic acid-based derivatives,ethers, ether-based derivatives, esters and ester-based derivatives,amines, amino-based derivatives, amides, amide-based derivatives,monocyclic or polycyclic arene, heteroarenes. arene-based derivatives,heteroarene-based derivatives, phenols, phenol-based derivatives,benzoic acid, and benzoic acid-based derivatives, and R₂ and R₄ may bethe same or different and are selected from the group consisting ofhydrogen, alkanes. cyclic alkanes, alkane-based derivatives, alkenes,cyclic alkenes, alkene-based derivatives, alkynes, alkyne-basedderivative, ketones, ketone-based derivatives, aldehydes, aldehyde-basedderivatives, carboxylic acids, carboxylic acid-based derivatives,ethers, ether-based derivatives, esters and ester-based derivatives,amines, amino-based derivatives, amides, amide-based derivatives,monocyclic or polycyclic arene, heteroarenes. arene-based derivatives,heteroarene-based derivatives, phenols, phenol-based derivatives,benzoic acid, and benzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises thegeneral formula:

wherein R₁, R₂, and R₃ may be the same or different and are selectedfrom the group consisting of hydrogen, carboxyl, alkanes. cyclicalkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes. arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, and benzoic acid-basedderivatives.

An exemplary and illustrative list of alkanes, cyclic alkanes, andalkane-based derivates are described herein. Non-limiting examples ofketones, ketone-based derivatives, aldehydes, aldehyde-basedderivatives; carboxylic acids, carboxylic acid-based derivatives,ethers, ether-based derivatives, esters, ester-based derivatives,amines, amino-based derivatives, amides, and amide-based derivatives arelisted herein. Exemplary monocyclic or polycyclic arene, heteroarenes,arene-based or heteroarene-based derivatives, phenols, phenol-basedderivatives, benzoic acid and benzoic acid-based derivatives aredescribed herein.

TABLE 1 Chemical names Formulas Methyl CH₃ Ethyl C₂H₅ Vinyl (ethenyl)C₂H₃ Ethynyl C₂H Cyclopropyl C₃H₅ Cyclobutyl C₄H₇ Cyclopentyl C₅H₉Cyclohexyl C₆H₁₁

TABLE 2 Chemical names Chemical formulas Acetonyl C₃H₅O Methanal(formaldehyde) CH₂O Paraldehyde C₆H₁₂O₃ Ethanoic acid CH₃COOH Diethylether C₄H₁₀O Trimethylamine C₃H₉N Acetamide C₂H₅NO Ethanol C₂H₅OHMethanol CH₃OH

TABLE 3 Chemical names Chemical formulas Benzol C₆H₆ Phenol C₆H₆OBenzoic acid C₇H₆O₂ Aniline C₆H₇N Toluene C₇H₈ Pyridazine C₄H₄N₂Pyrimidine C₄H₄N₂ Pyrazine C₄H₄N₂ Biphenyl C₁₂H₁₀The compositions of the present invention and any functionally activederivatives thereof may be obtained by any suitable means. In specificembodiments, the derivatives of the invention are provided commercially,although in alternate embodiments the derivatives are synthesized. Thechemical synthesis of the derivatives may employ well known techniquesfrom readily available starting materials. Such synthetictransformations may include, but are not limited to protection,de-protection, oxidation, reduction, metal catalyzed C—C cross coupling,Heck coupling or Suzuki coupling steps (see for example, March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structures, 5′Edition John Wiley and Sons by Michael B. Smith and Jerry March,incorporated here in full by reference).

V. Embodiments for Targeting Stat3

STAT proteins, of which there are seven (1, 2, 3, 4, 5A, 5B and 6),transmit peptide hormone signals from the cell surface to the nucleus.Detailed structural information of STAT proteins currently is limited toStat1 and Stat3. Stat1 was the first STAT to be discovered (Fu et al.,1992) and is required for signaling by the Type I and II IFNs (Meraz etal, 1996; Wiederkehr-Adam et al, 2003; Durbin et al, 1996; Haan et al.,1999). Studies in Stat1-deficient mice (Meraz et al, 1996; Durbin et al,1996; Ryan et al., 1998) support an essential role for Stat1 in innateimmunity, notably against viral pathogens. In addition, Stat1 is apotent inhibitor of growth and promoter of apoptosis (Bromberg andDarnell, 2000). Also, because tumors from carcinogen-treated wild-typeanimals grow more rapidly when transplanted into the Stat1-deficientanimals than they do in a wild-type host, Stat1 contributes to tumorsurveillance (Kaplan et al., 1998).

Stat3 was originally termed acute-phase response factor (APRF) becauseit was first identified as a transcription factor that bound toIL-6-response elements within the enhancer-promoter region of variousacute-phase protein genes (Akira, 1997). In addition to receptors forthe IL-6 cytokine family, other signaling pathways are linked to Stat3activation include receptors for other type I and type II cytokinereceptors, receptor tyrosine kinases, G-protein-coupled receptors andSrc kinases (Schindler and Darnell, 1995; Turkson et al., 1998).Targeted disruption of the mouse Stat3 gene leads to embryonic lethalityat 6.5 to 7.5 days (Takeda et al., 1997) indicating that Stat3 isessential for early embryonic development possibly gastrulation orvisceral endoderm function (Akira, 2000). Tissue-specific deletion ofStat3 using Cre-lox technology has revealed decreased mammary epithelialcell apoptosis resulting in delayed breast involution during weaning(Chapman et al., 1999). Recent findings indicate that switching of thepredominant STAT protein activated by a given receptor can occur when aSTAT downstream of that receptor is genetically deleted (Costa-Pereiraet al., 2002; Qing and Stark, 2004). These findings suggest thepossibility that the effect of Stat3 deletion in breast tissue may bemediated indirectly by increased activation of other STAT proteins,especially Stat5.

Stat1 and Stat3 isoforms. Two isoforms of Stat1 and Stat3 have beenidentified—α (p91 and p92, respectively) and β (p84 and p83,respectively) (Schindler et al., 1992; Schaefer et al., 1995;Caldenhoven et al., 1996; Chakraborty et al., 1996)—that arise due toalternative mRNA splicing (FIG. 13). In contrast to Stat1β (712 aa), inwhich the C-terminal transactivation is simply deleted, the 55 aminoacid residues of Stat3α are replaced in Stat3 β by 7 unique amino acidresidues at its C-terminus. Unlike Stat1 β, Stat3 β is not simply adominant-negative of Stat3α (Maritano et al., 2004) and regulates genetargets in a manner distinct from Stat3 β (Maritano et al., 2004; Yoo etal., 2002). Stat3α has been demonstrated to contribute to transformationin cell models and many human cancers including breast cancer. Stat3αwas shown to be constitutively activated in fibroblasts transformed byoncoproteins such as v-Src (Yu et al., 1995; Garcia and Jove, 1998) andto be essential for v-Src-mediated transformation (Turkson et al., 1998;Costa-Pereira et al., 2002). In contrast to Stat3α, Stat3β antagonizedv-Src transformation mediated through Stat3α (Turkson et al., 1998).Overexpression of a constitutively active form of Stat3α in immortalizedrat or mouse fibroblasts induced their transformation and conferred theability to form tumors in nude mice (Bromberg et al., 1999). Stat3 hasbeen shown to be constitutively activated in a variety of hematologicaland solid tumors including breast cancer (Dong et al., 2003; Redell andTweardy, 2003) as a result of either autocrine growth factor productionor dysregulation of protein tyrosine kinases. In virtually all cases,the isoform demonstrating increased activity is Stat3α.

Targeting Stat3α while sparing Stat1. Given its multiple contributoryroles to oncogenesis, Stat3 has recently gained attention as a potentialtarget for cancer therapy (Bromberg, 2002; Turkson, 2004). While severalmethods of Stat3 inhibition have been employed successfully and haveestablished proof-of-principle that targeting Stat3 is potentiallybeneficial in a variety of tumor systems including breast cancer inwhich Stat3 is constitutively activated (Epling-Burnette et al., 2001;Yoshikawa et al., 2001; Li and Shaw, 2002; Catlett-Falcone et al., 1999;Mora et al., 2002; Grandis et al., 2000; Leong et al., 2003; Jing etal., 2003; Jing et al., 2004; Turkson et al., 2001; Ren et al., 2003;Shao et al., 2003; Turkson et al., 2004; Uddin et al., 2005); all havepotential limitations for translation to clinical use for cancer therapyrelated to issues regarding delivery, specificity or toxicity.

Specific strategies that target Stat3 by identifying inhibitors of Stat3recruitment and/or dimerization have been pursued by several groups(Turkson et al., 2001; Ren et al., 2003; Shao et al., 2003; Uddin etal., 2005; Song et al., 2005; Schust et al., 2006). As outlined below,this strategy has the potential to achieve specificity based on theobservation that the preferred pY peptide motif of each STAT protein isdistinct. When coupled to a small molecule approach, this strategy hasthe potential to overcome issues of delivery and toxicity.

Targeting Stat3α while sparing Stat3β. Some of the distinct biochemicalfeatures of Stat3β vs. Stat3α, notably constitutive activation and a10-to-20 fold increased DNA binding affinity, have been attributed tothe absence of the C-terminal transactivation domain (TAD) resulting inincreased Stat3β dimer stability (Park et al., 1996; Park et al., 2000).Increased dimer stability likely results from higher binding affinity ofthe SH2 domain to pY peptide motifs when in the context of Stat3βcompared to Stat3α because of reduced steric hindrance conferred byremoval of the TAD. These differential biochemical features betweenStat3 α and Stat3β are exploited to develop a chemical compound thatselectively targets Stat3 α, in some embodiments. This selectivityenhances the anti-tumor effect of such compounds, in certain cases,because they would spare Stat3β, which functions to antagonize theoncogenic functions of Stat3α.

In certain embodiments of the invention, specific therapies targetingStat3 signaling are useful for treatment of cachexia.

VI. Combination Therapy

It is an aspect of this invention that a composition as disclosed hereinis used in combination with another agent or therapy method, such asanother muscle wasting and/or muscle weakness and/or cachexia treatmentand/or a treatment for an underlying condition. The composition(s)(which may or may not be a Stat3 inhibitor) may precede or follow theother agent treatment by intervals ranging from minutes to weeks, forexample. In embodiments where the other agent and the composition of theinvention are applied separately to an individual with cachexia, such asupon delivery to an individual suspected of having cachexia, known tohave cachexia, or at risk for having cachexia, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the agent and composition of the inventionwould still be able to exert an advantageously combined effect on theindividual.

For example, in such instances, it is contemplated that one may contactthe cell, tissue or organism with one, two, three, four or moremodalities substantially simultaneously (i.e., within less than about aminute) with the composition of the invention. In other aspects, one ormore agents may be administered within about 1 minute, about 5 minutes,about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes,about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours,about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours,about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours,about 46 hours, about 47 hours, to about 48 hours or more prior toand/or after administering the composition of the invention. In certainother embodiments, an agent may be administered within of from about 1day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 7 days, about 8 days, about 9 days, about 10 days, about 11days, about 12 days, about 13 days, about 14 days, about 15 days, about16 days, about 17 days, about 18 days, about 19 days, about 20, to about21 days prior to and/or after administering the composition of theinvention, for example. In some situations, it may be desirable toextend the time period for treatment significantly, such as whereseveral weeks (e.g., about 1, about 2, about 3, about 4, about 5, about6, about 7 or about 8 weeks or more) lapse between the respectiveadministrations. In some situations, it may be desirable to extend thetime period for treatment significantly, such as where several months(e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 orabout 8 weeks or more) lapse between the respective administrations.

Various combinations may be employed, the composition of the inventionis “A” and the secondary agent, which can be any other cancertherapeutic agent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapeutic compositions of the present inventionto a patient will follow general protocols for the administration ofdrugs, taking into account the toxicity. It is expected that thetreatment cycles would be repeated as necessary.

In embodiments wherein an individual has cachexia associated withcancer, the individual may also be receiving chemotherapy,immunotherapy, hormone therapy, radiation therapy and/or surgery.

VII. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise aneffective amount of a composition as disclosed herein dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical” or “pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one Stat3 inhibitor of the invention,and in some cases an additional active ingredient, will be known tothose of skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The composition(s) may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it needs to be sterile for such routes of administration such asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, in lipid compositions (e.g., liposomes), as an aerosol, or byother method or any combination of the forgoing as would be known to oneof ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an individual can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, and the route of administration. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of a composition. In other embodiments, theactive compound may comprise between about 2% to about 75% of the weightof the unit, or between about 25% to about 60%, for example, and anyrange derivable therein. In other non-limiting examples, a dose may alsocomprise from about 0.1 mg/kg/body weight, 0.5 mg/kg/body weight, 1mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/bodyweight, about 20 mg/kg/body weight, about 30 mg/kg/body weight, about 40mg/kg/body weight, about 50 mg/kg/body weight, about 75 mg/kg/bodyweight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about350 mg/kg/body weight, about 500 mg/kg/body weight, about 750 mg/kg/bodyweight, to about 1000 mg/kg/body weight or more per administration, andany range derivable therein. In non-limiting examples of a derivablerange from the numbers listed herein, a range of about 10 mg/kg/bodyweight to about 100 mg/kg/body weight, etc., can be administered, basedon the numbers described above. In certain embodiments of the invention,various dosing mechanisms are contemplated. For example, the compositionmay be given one or more times a day, one or more times a week, or oneor more times a month, and so forth.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including, but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The composition may be formulated in a free base, neutral or salt form.Pharmaceutically acceptable salts include the salts formed with the freecarboxyl groups derived from inorganic bases such as for example,sodium, potassium, ammonium, calcium or ferric hydroxides; or suchorganic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample, liquid polyol or lipids; by the use of surfactants such as, forexample, hydroxypropylcellulose; or combinations thereof such methods.In many cases, it will be preferable to include isotonic agents, suchas, for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the instantinvention in the required amount of the appropriate solvent with variousamounts of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

VIII. Kits of the Invention

Any of the compositions described herein may be comprised in a kit, andthey are housed in a suitable container. The kits will thus comprise, insuitable container means, one or more compositions and, in some cases,an additional agent of the present invention. In some cases, there areone or more agents other than the composition of the disclosure that areincluded in the kit, such as one or more other agents for the treatmentof muscle wasting and/or muscle weakness and/or cachexia and/or one ormore agents for the treatment of an underlying condition associated withmuscle wasting and/or muscle weakness and/or cachexia. In particularembodiments, there is an apparatus or any kind of means for thediagnosing of muscle wasting and/or muscle weakness and/or cachexia.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the composition, additional agent, and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained.

Compositions may also be formulated into a syringeable composition. Inwhich case, the container means may itself be a syringe, pipette, and/orother such like apparatus, from which the formulation may be applied toan infected area of the body, injected into an animal, and/or evenapplied to and/or mixed with the other components of the kit. However,the components of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Materials and Methods

Virtual ligand screening. The inventors isolated the three-dimensionalstructure of the Stat3 SH2 domain from the core fragment structure ofphosphorylated Stat3 homodimers bound to DNA (Becker et al., 1998)deposited in the RCSB Protein Data Bank (PDB) databank (PDB code 1BG1)and converted it to be an Internal Coordinate Mechanics (ICM)-compatiblesystem by adding hydrogen atoms, modifying unusual amino acids, makingcharge adjustments and performing additional cleanup steps. In addition,the inventors retrieved the coordinates of the Stat1 SH2 domain from thePDB databank (PDB code 1BF5) for use in computational selectivityanalysis (Chen et al., 1998). Commercial chemical databases (Chembridge,Asinex, ChemDiv, Enamine, Keyorganics and Life Chemicals) were chosen assources of compounds for screening in silico. Selection was of the amidehydrogen of E638 within the site that binds the +3 residue (Q, C or T)within the pY-peptide ligand (Shao et al., 2006) as the central point ofthe binding pocket, which consisted of a cube with dimensions16.0×16.9×13.7 angstrom. In addition to the +3 binding site, this cubecontained the pY residue binding site consisting mainly of R609 and K591(Shao et al., 2006) and a hydrophobic binding site consisting ofLoop_(βC-βD) and Loop_(αB-αC). Sequence alignment and overlay of theStat3 and Stat1 structures revealed substantial differences in sequenceof these loops; lack of their superimposition indicated that this regionmight serve as a selectivity filter (Cohen et al., 2005). A flexibledocking calculation (Totrov and Abagyan 1997) was performed in order todetermine the global minimum energy score and thereby predict theoptimum conformation of the compound within the pocket. A compound wasselected for purchase and biochemical testing based on fulfilling thecriteria of interaction analysis (CIA): 1) global minimum energy score5-30, 2) formation of a salt-bridge and/or H-bond network within thepY-residue binding site and 3) formation of a H-bond with or blockingaccess to the amide hydrogen of E638. Most, but not all, compounds alsointeracted with the hydrophobic binding site.

Stat3 SH2/pY-peptide binding assay. Stat3 binding assays were performedat 25° C. with a BIAcore 3000 biosensor using 20 mM Tris buffer pH 8containing 2 mM mercaptoethanol and 5% DMSO as the running buffer (Kimet al., 2005). Phosphorylated and control non-phosphorylatedbiotinylated EGFR derived dodecapeptides based on the sequencesurrounding Y1068 (Shao et al., 2004) were immobilized on a streptavidincoated sensor chip (BIAcore inc., Picataway N.J.). The binding of Stat3was conducted in 20 mM Tris buffer pH 8 containing 2 mMß-mercaptoethanol at a flow rate of 10 uL/min for 1-2 minute. Aliquotsof Stat3 at 500 nM were premixed with compound to achieve a finalconcentration of 1-1,000 uM and incubated at 4° C. prior to beinginjected onto the sensor chip. The chip was regenerated by injecting 10uL of 100 mM glycine at pH 1.5 after each sample injection. A control(Stat3 with DMSO but without compound) was run at the beginning and theend of each cycle (40 sample injections) to ensure that the integrity ofthe sensor chip was maintained throughout the cycle run. The average ofthe two controls was normalized to 100% and used to evaluate the effectof each compound on Stat3 binding. Responses were normalized by dividingthe value at 2 min by the response obtained in the absence of compoundsat 2 min and multiplying by 100. IC₅₀ values were determined by plotting% maximum response as a function of log concentration of compound andfitting the experimental points to a competitive binding model using afour parameter logistic equation:R=R_(high)−(R_(high)−R_(low))/(1+conc/A1){circumflex over ( )}A2, whereR=percent response at inhibitor concentration, R_(high)=percent responsewith no compound, R_(low)=percent response at highest compoundconcentration, A2=fitting parameter (slope) and A1=IC₅₀ (BIAevaluationSoftware version 4.1).

Immunoblot assay. The human hepatocellular carcinoma cell line (HepG2)was grown in 6-well plates under standard conditions. Cells werepretreated with compounds (0, 1, 3, 10, 30, 100 and 300 uM) for 1 hourthen stimulated under optimal conditions with either interferon gamma(IFN-γ; 30 ng/ml for 30 min) to activate Stat1 or interleukin-6 (IL-6;30 ng/ml for 30 min) to activate Stat3 (30-31). Cultures were thenharvested and proteins extracted using high-salt buffer, as described(Shao et al., 2006). Briefly, extracts were mixed with 2× sodium dodecylsulfate (SDS) sample buffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20%glycerol; 10%2-mercaptoethanol) at a 1:1 ratio and heated for 5 minutesat 100° C. Proteins (20 μg) were separated by 7.5% SDS-PAGE andtransferred to polyvinylidene fluoride (PVDF) membrane (Millipore,Waltham, Mass.) and immunoblotted. Prestained molecular weight markers(Biorad, Hercules, Calif.) were included in each gel. Membranes wereprobed serially with antibody against Stat1 pY⁷⁰¹ or Stat3 pY⁷⁰⁵followed by antibody against Stat1 or Stat3 (Transduction labs,Lexington, Ky.) then antibody against β-actin (Abcam, Cambridge, Mass.).Membranes were stripped between antibody probing using Restore™ WesternBlot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, Mass.)per the manufacturer's instructions. Horseradish peroxidase-conjugatedgoat-anti-mouse IgG was used as the secondary antibody (InvitrogenCarlsbad, Calif.) and the membranes were developed with enhancedchemiluminescence (ECL) detection system (Amersham Life Sciences Inc.;Arlington Heights, Ill.).

Similarity screen. Three compounds identified in the initial virtualligand screening (VLS) Cpd3, Cpd30 and Cpd188 inhibited Stat3SH2/pY-peptide binding and IL-6-mediated Stat3 phosphorylation and werechosen as reference molecules for similarity screening. A fingerprintsimilarity query for each reference compound was submitted toMolcart/ICM (Max Distance, 0.4). Similarity between each referencemolecule and each database molecule was computed and the similarityresults were ranked in decreasing order of ICM similarity score (Eckertand Bajorath 2007). The databases searched included ChemBridge,LifeChemicals, Enamine, ChemDiv, Asinex, AcbBlocks, KeyOrganics andPubChem for a total of 2.47 million compounds. All compounds identifiedwere docked into the binding pocket of Stat3 SH2 domain in silico.Compounds that fulfilled CIA criteria were purchased and tested asdescribed for compounds identified in the primary screen.

Electrophoretic Mobility Shift Assay (EMSA): EMSA was performed usingthe hSIE radiolabeled duplex oligonucleotide as a probe as described(Tweardy et al., 1995). Briefly, high salt extracts were prepared fromHepG2 cells incubated without or with IL-6 (30 ng/ml) for 30 minutes.Protein concentration was determined by Bradford Assay and 20 ug ofextract was incubated with compound (300 uM) for 60 minutes at 37° C.Bound and unbound hSIE probe was separated by polyacrylamide gelelectrophoresis (4.5%). Gels were dried and autoradiographed.

Molecular modeling. All 3-D configurations of the Stat3 SH2 domaincomplexed with compounds were determined by global energy optimizationthat involves multiple steps: 1) location of organic molecules wereadjusted as a whole in 2 Å amplitude by pseudo-Brownian randomtranslations and rotations around the molecular center of gravity, 2)the internal variables of organic molecules were randomly changed. 3)coupled groups within the Stat3 SH2 domain side-chain torsion angleswere sampled with biased probability shaking while the remainingvariables of the protein were fixed, 4) local energy minimizations wereperformed using the Empirical Conformation Energy Program for Peptidestype-3 (ECEPP3) in a vacuum (Nemethy et al., 1992) withdistance-dependent dielectric constant ε=4r, surface-based solventenergy and entropic contributions from the protein side chains evaluatedadded and 5) conformations of the complex, which were determined byMetropolis criteria, were selected for the next conformation-scanningcircle. The initial 3-dimensional configuration of the Stat1 SH2 domainin a complex with each compound was predicted and generated bysuperimposing, within the computational model, the 3-dimensionalfeatures of the Stat1 SH2 onto the 3-dimensional configuration of theStat3 SH2 domain in a complex with each compound. The finalcomputational model of Stat1 SH2 in a complex with each compound wasdetermined by local minimization using Internal Coordinate Force Field(ICFF)-based molecular mechanics (Totrov and Abagyan 1997). Theinventors computed the van der Waals energy of the complex of Stat1 or3-SH2 bound with each compound using Lennard-Jones potential withECEPP/3 force field (Nemethy et al., 1992).

Confocal and high-throughput fluorescence microscopy. Confocal andhighthroughput fluorescence microscopy (HTFM) of MEF/GFP-Stat3c cellswere performed as described (Huang et al., 2007). Briefly, for confocalfluorescence microscopy, cells were grown in 6-well plates containing acover slip. For HTFM, cells were seeded into 96-well CC3 plates at adensity of 5,000 cells/well using an automated plating system. Cellswere cultured under standard conditions until 85-90% confluent. Cellswere pretreated with compound for 1 hour at 37° C. then stimulated withIL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes. Cells were fixedwith 4% formaldehyde in PEM Buffer (80 mM Potassium PIPES, pH 6.8, 5 mMEGTA pH 7.0, 2 mM MgCl₂) for 30 minutes at 4° C., quenched in 1 mg/ml ofNaBH4 (Sigma) in PEM buffer and counterstained for 1 min in4,6-diamidino-2-phenylindole (DAPI; Sigma; 1 mg/ml) in PEM buffer. Coverslips were examined by confocal fluorescent microscopy. Plates wereanalyzed by automated HTFM using the Cell Lab IC Image Cytometer (IC100)platform and CytoshopVersion 2.1 analysis software (Beckman Coulter).Nuclear translocation is quantified by using the fraction localized inthe nucleus (FLIN) measurement (Sharp et al., 2006).

Example 2 Identification by VLS of Compounds that Blocked Stat3 Bindingto its Phosphopeptide Ligand and Inhibited IL-6-Mediated Phosphorylationof Stat3

The VLS protocol was used to evaluate a total of 920,000 drug-likecompounds. Of these, 142 compounds fulfilled CIA criteria. Thesecompounds were purchased and tested for their ability to block Stat3binding to its phosphopeptide ligand in a surface plasmon resonance(SPR)-based binding assay and to inhibit IL-6-mediated phosphorylationof Stat3. SPR competition experiments showed that of the 142 compoundstested, 3 compounds Cpd3, Cpd30 and Cpd188-were able to directly competewith pY-peptide for binding to Stat3 with IC₅₀ values of 447, 30, and 20μM, respectively (FIGS. 1 and 3; Table 4).

TABLE 4 IC₅₀ values (μM) of 6 active compounds Assay Cpd3 Cpd30 Cpd188Cpd3-2 Cpd3-7 Cpd30-12 SPR 447¹ 30 20 256 137 114 pStat3  91  18 73 14463 60 HTM 131  77 39 150 20 >300 ¹Data presented are the mean or mean ±SD; ND = not determined.

In addition, each compound inhibited IL-6-mediated phosphorylation ofStat3 with IC50 values of 91, 18 and 73 μM respectively (FIGS. 2A-2F;Table 4).

Similarity screening with Cpd3, Cpd30 and Cpd188 identified 4,302additional compounds. VLS screening was performed with each of thesecompounds, which identified 41 compounds that fulfilled CIA criteria;these were purchased and tested. SPR competition experiments showed thatof these 41 compounds, 3 compounds-Cpd3-2, Cpd3-7 and Cpd30-12-were ableto directly compete with pY-peptide for binding to Stat3 with IC₅₀values of 256, 137 and 114 μM, respectively (FIGS. 1 and 3; Table 4). Inaddition, each compound inhibited IL-6-mediated phosphorylation of Stat3with IC50 values of 144, 63 and 60 μM, respectively (FIGS. 2A-2F; Table4).

Example 3 Compound-Mediated Inhibition of Ligand-StimulatedPhosphorylation of Stat3 is Specific for Stat3 Vs. Stat1

While Stat3 contributes to oncogenesis, in part, through inhibition ofapoptosis, Stat1 is anti-oncogenic; it mediates the apoptotic effects ofinterferons and contributes to tumor surveillance (Kaplan et al., 1998;Ramana et al., 2000). Consequently, compounds that target Stat3 whilesparing Stat1, leaving its anti-oncogenic functions unopposed, mayresult in a synergistic anti-tumor effect. To assess the selectivity ofthe compounds for Stat3 vs. Stat1, HepG2 cells were incubated with Cpd3,Cpd30, Cpd188, Cpd3-2, Cpd3-7, and Cpd30-12 (300 μM) for 1 hour at 37°C. before IFN-γ stimulation (FIG. 4). Only treatment with Cpd30-12blocked Stat1 phosphorylation while each of the other fivecompounds-Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7-did not. Thus, five ofthe six exemplary compounds identified were selective and inhibitedligand-stimulated phosphorylation of Stat3 but not Stat1.

Example 4 Sequence Analysis and Molecular Modeling of the Interaction ofEach Compound with the Stat3 Vs. Stat1 SH2 Domain

To understand at the molecular level the basis for the selectivity ofCpds 3, 30, 188, 3-2 and 3-7 and the absence of selectivity in the caseof Cpd 30-12, the amino acid sequence and available structures of theStat1 and Stat3 SH2 domain were compared and also it was examined howeach compound interacted with both. Sequence alignment revealed identityin the residues within Stat3 and Stat1 corresponding to the binding sitefor the pY residue and the +3 residue (FIG. 5A). In addition, overlay ofthe Stat3 and Stat1 SH2 structures revealed that the loops thatcontained these binding sites were superimposed (FIG. 5B). In contrast,sequence alignment revealed substantial differences in the sequence ofthe regions of the SH2 domain corresponding to the loops forming thehydrophobic binding site (FIG. 5A). In addition, review of the overlayof Stat3 and Stat1 SH2 domains revealed that, in contrast to the closeapposition of the two loops of Stat3 that form the hydrophobic bindingsite, the corresponding two loops of Stat1 are not closely apposed toform a pocket (FIG. 5B).

Review of computational models of Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7in a complex with the Stat3 SH2 domain revealed that each hassignificant interactions with the Stat3 SH2 domain binding pocket at allthree binding sites, the pY-residue binding site, the +3 residue bindingsite and the hydrophobic binding site (FIGS. 6A, B, C, D, and E). Incontrast, Cpd30-12 interacts with the pY-residue binding site and blocksaccess to the +3 residue-binding site but does not interact with orblock access to the hydrophobic binding site (FIG. 6F). In addition, vander Waals energies of the 5 selective compounds were much more favorablefor their interaction with the loops of Stat3 forming the hydrophobicbinding site than with corresponding loops of Stat1 (FIG. 5C). Thus,computer modeling indicated that activity of compounds against Stat3derives from their ability to interact with the binding sites for the pYand the +3 residues within the binding pocket, while selectivity forStat3 vs. Stat1 derives from the ability of compounds to interact withthe hydrophobic binding site within the Stat3 SH2 binding pocket, whichserved as a selectivity filter.

Example 5 Inhibition of Nuclear Translocation of Phosphorylated Stat3 byCpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7 Assessed by HTFM

Following its phosphorylation on Y705, Stat3 undergoes a change inconformation from head-to-head dimerization mediated through itsN-terminal oligomerization domain to tail-to-tail dimerization mediatedby reciprocal SH2/pY705-peptide ligand interactions. This conformationalchange is followed by nuclear accumulation. Compounds that targetedSH2/pY-peptide ligand interactions of Stat3 would be expected to inhibitnuclear accumulation of Stat3. To determine if this was the case withthe compounds herein, a nuclear translocation assay (FIGS. 7A-7B) wasemployed using murine embryonic fibroblast (MEF) cells that aredeficient in endogenous Stat3 but constitutively express GFP-taggedStat3α at endogenous levels, MEF/GFP-Stat3 α (Huang et al., 2007).Preincubation of MEF/GFP-Stat3 α cells with Cpd3, Cpd30, Cpd188, Cpd3-2and Cpd3-7, but not Cpd30-12, blocked ligand-mediated nucleartranslocation of GFP-Stat3 α with IC₅₀ values of 131, 77, 39, 150 and 20μM (FIGS. 7A-7B and Table 4).

Example 6 Destabilization of Stat3-DNA Complexes by Cpd3 and Cpd3-7

Once in the nucleus, Stat3 dimers bind to specific DNA elements toactivate and, in some instances, repress gene transcription.Tyrosine-phosphorylated dodecapeptides based on motifs within receptorsthat recruit Stat3 have previously been shown to destabilize Stat3(Chakraborty et al., 1999; Shao et al., 2003). Compounds that bind tothe phosphopeptide-binding site of Stat3 might be expected to do thesame. To determine if this was the case for any of the identifiedcompounds, extracts of IL-6-stimulated HepG2 cells were incubated inbinding reactions containing radiolabeled hSIE (FIG. 8) and each of thefive selective compounds (300 M). Incubation with Cpd3 or Cpd3-7 reducedthe amount of hSIE shifted by half or greater. The other compounds didnot have a detectable effect on the Stat3:hSIE band intensity. Thus, 2of the 5 selective compounds destabilized Stat3:hSIE complexes.

Example 7 Exemplary Approach for Stat3 Inhibitors for Cancer Stem Cells

In the field of Stat3 probe development the inventors have focused onsmall molecule Stat3 probes (Xu et al., 2009), and several features ofthe small molecule program are useful, including: 1) a clearly definedmode of action of these probes: they target the Stat3 Src-homology (SH)2 domain that is involved in 2 steps in the Stat3 activation pathway; 2)their specificity of action; and 3) the potential for using lead probesidentified so far to identify probes with 2-to-3 logs greater activitybased on recent and exemplary SAR analysis and medicinal chemistryconsiderations outlined below.

In specific embodiments, compound affinity is improved upon gaining alog greater affinity upon moving from 1^(st) generation to 2ndgeneration probes using 3-D pharmacophore analysis. In addition,selectivity is improved through modeling embodiments, in particularthrough identification of a distinct hydrophobic binding domain in thephosphopeptide binding pocket of Stat3 SH2 vs. the Stat1 SH2 (Xu et al.,2009).

Identification of 1st generation Stat3 chemical probes. To developchemical probes that selectively target Stat3, the inventors virtuallyscreened 920,000 small drug-like compounds by docking each into thepeptide-binding pocket of the Stat3 SH2 domain, which consists of threesites—the pY-residue binding site, the +3 residue-binding site and ahydrophobic binding site, which served as a selectivity filter (Xu etal., 2009). Three compounds (Cpd3, Cpd30 and Cpd188) satisfied criteriaof interaction analysis, competitively inhibited recombinant Stat3binding to its immobilized pY-peptide ligand and inhibited IL-6-mediatedtyrosine phosphorylation of Stat3. These compounds were used in asimilarity screen of 2.47 million compounds, which identified 3 morecompounds (Cpd3-2, Cpd3-7 and Cpd30-12) with similar activities.Examinations of the 6 active compounds for the ability to inhibitIFN-γ-mediated Stat1 phosphorylation revealed that all but Cpd30-12 wereselective for Stat3. Molecular modeling of the SH2 domains of Stat3 andStat1 bound to compound revealed that compound interaction with thehydrophobic binding site was the basis for selectivity. All 5 selectivecompounds inhibited nuclear-tocytoplasmic translocation of Stat3, while3 of 5 compounds (Cpd3, Cpd30 and Cpd188) induced apoptosispreferentially of exemplary breast cancer cell lines with constitutiveStat3 activation.

Identification of 2nd generation Stat3 chemical probes. The similarityscreening described above did not yield any hits using Cpd188, the mostactive of the 3 lead compounds, as the query compound. Consequently, theinventors repeated 2-D similarity screening using the scaffold of Cpd188as the query structure and the Life Chemicals library, which yielded 207hits. 3-D pharmacophore analysis was performed on these 207 compoundsusing Ligand Scout and the top 39 scoring compounds were purchased andtested for inhibition of Stat3 binding to its phosphopeptide ligand bySPR. All but six of these 39 compounds have measurable SPR IC50s, with19 having IC50 values equal to or less than the parent compound and 2(Cpd188-9 and Cpd188-15) having IC50 values one log lower. Examinationof these 19 compounds has revealed a statistically significantcorrelation between 3-D pharmacophore scores and SPR IC50s and as wellas 3-D pharmacophore score and IC50s for inhibition of ligand-mediatedcytoplasmic-to-nuclear translocation. In addition, both Cpd188-9 andCpd188-15 exhibited a log greater activity in inducing human leukemiccell line apoptosis than the parent Cpd188 (FIG. 15). In addition,Cpd188-38 exhibited a 2 logs greater activity than parent Cpd188 ininhibiting cytoplasmic-to-nuclear translocation in HTFM assay, whileCpd188-15 exhibited a 1 log greater activity than parent Cpd188 indecreasing MSFE (Table 5). Furthermore, several of the second-generation188-like compounds represent a substantial improvement over Cpd188 froma medicinal chemistry, metabolism and bioavailability standpoint. Inparticular, Cpd188-9 lacked both carboxyl groups, which in particularcases improves cell permeability and/or the thioether group, which issubject to oxidation. R2=0.2 P=0.013 (M)

TABLE 5 Summary of Certain 2^(nd) Generation 188-like Compounds SPRIC₅₀, HTFM IC₅₀, Mammosphere Compound μM* μM* ~IC₅₀, μM*** 188 20** 32 ±4   30-100 188-1 6 ± 2 26 ± 4  30 188-9 3 ± 2 47 ± 21 10 188-10 8 ± 3 22± 19 30 188-15 2 ± 1 49 3 188-16 4 ± 0 9 ± 5 30 188-17 4 ± 2 76 30188-18 4 ± 1 27 ± 8  30 188-38 19 ± 9  0.4 ± 0.1 10-30 *mean ± SD **Xuet al PLos ONE ***SUM159PT and HS578T cells plated (6 wells per test)without or with compound at 1, 10, or 100 μM, incubated 3 d; spherescounted on day 3

Structure-activity relationship (SAR) analysis of 2nd generation Stat3probes. All of the 39 second generation compounds described above, plusCpd188 itself, are derivatives of N-naphth-1-yl benzenesulfamide. Uponcareful analysis of their structure-activity relationships (SAR), theinventors found that most of these Cpd188-like compounds (38 out of 40:the rest of 2 are weak and will be described below in EXP ID) can bedivided into three structural groups in a general trend of decreasedactivity, as shown in FIG. 16. Five compounds in Group III are actuallythe parents of compounds in Groups I and II. Addition of a variety ofgroups (the —R group highlighted in red in the general structure ofGroup I in FIG. 16), such as a triazole-3-yl-mercapto (188-15) or achloro (188-10) group, to the 3-position of the naphthylamine ring ledto the Group I compounds, which are the most potent series of Stat3probes. In a specific embodiment, this is the most important contributorto the inhibitory activity: a total of eight 3-substituents are found inGroup I compounds, which invariably enhance the activity by severalorders of magnitude.

Most Stat3 probes in Group II contain a 5-membered ring that combinesthe 3-R and 4-OR2 groups, such as a furan (188-11). However, thecompounds in this group are, in average, ˜5× less active than the GroupI compounds, which indicates that in certain aspects the H atom of the4-hydroxy group (highlighted in blue in the general structure of Group Iin FIG. 16) is important, e.g., involved in a favorable H-bond with theprotein. Lacking the ability to form the H-bond attributes to the weakeractivities of Group II probes, in particular cases. These considerationsunderlie a medicinal chemistry approach outlined below.

Example 8 Medicinal Chemistry for Synthesis of 3^(RD) Generation188-Like Sulfamide Stat3 Probes

The crystal structure of Stat3 shows that the SH2 domain has a large,widely dispersed and generally shallow binding area with several valleysand hills that recognize the pY-peptide ligand (FIG. 18).Structure-based molecular modeling (docking) was useful in identifyingthe contribution of the hydrophobic binding surface of the Stat3 SH2domain as a selectivity filter (Xu et al., 2009). However, differentdocking programs gave distinct binding poses for the same probe over thebinding surface with similar predicted binding affinities. The inventorstherefore in particular embodiments, based on initial SAR resultsoutlined above, use traditional medicinal chemistry to further carry outan exemplary comprehensive structure activity relationship study, tooptimize the activity as well as the selectivity of this novel class ofsulfamide probes of Stat3. Compound 188-15 serves as a scaffold formaking the new generation compounds, as shown schematically (FIG. 16).

In addition, chemistry for making these compounds is straightforwardwith a good yield, involving the reaction of a sulfonyl chloride with ananiline/amine, which can be either obtained commercially or synthesizedreadily.

For the proposed modifications described below, one can consult FIG. 17.EXP IA. Modification 1. Since almost all of the 2^(nd) generation probescontain a phenylsulfonyl group, the first step towards activityoptimization focuses on synthesizing a series of compounds that have alarger (e.g., bicyclic or tricyclic) or an alkyl sulfonyl group. Thegeneral synthetic route is shown as follows:

There are about 4,300 commercially available sulfonyl chlorides, amongwhich 25, such as those shown above, are selected to make probes.Aniline 2, which is the amine component of compound 188-10 (FIG. 16),one the most active probes, is readily made in a simple two stepreaction from nitro compound 1. One can first make 25 (for example)compounds and test their activities in an in vitro rapid throughput SPRand in vivo HTFM assays. Based on the outcomes of structure-activityrelationship study, more compounds can be designed and synthesized andtested in an iterative manner until optimization of this modification.

EXP IB. Modification 2. Next, one can modify the 3-substituent of thenaphthylamine ring, based on either the structure of compound 188-15,for example. Prior SAR studies demonstrated this substituent is usefulto the activity of this class of probes, in certain embodiments.However, a total of 8 groups at this position with a huge difference insize, from a single atom Cl to a large, bicyclicbenzothiazole-2-ylmercapto group, showed similar activities. Thisfeature indicates that in certain embodiments modifications at thisposition should be more focused on other properties, such aselectrostatic interactions with the protein, as exemplified below. Inaddition, many of these groups are thioethers, which may be subjected tooxidation/degradation in vivo and lead to an unfavorable pharmacokineticprofile, in particular aspects. The central —S— atom is changed to amore metabolically stable isosteres, such as —CH₂—, —NH—, and —O—, incertain cases. In certain aspects one can synthesize the followingcompounds to optimize the 3-substituent:

The synthesis is also started from 1, in certain cases. Regio-selectivehalogenation and formylation at the 3-position gives rise to twocompounds, i.e., bromo- or iodo-compound 3 and aldehyde 4, which areversatile, common starting compounds for introducing a wide range ofsubstituents at this position (e.g., those listed above).

Moreover, the crystal structure of Stat3 SH2 domain also provides strongevidence that more compounds with different electrostatic properties areuseful for characterization. The electrostatic molecular surface of theprotein shows two distinct features, as shown in FIG. 18. The first oneis the negatively charged Glu638 surface stands out in the center. Next,of particular interest is a positively charged area, composed of Arg609and Lys591 located in the edge of the domain, which is actually the pY(phosphorylated tyrosine) binding site of the receptor. The inventorsalso found that introducing a negatively charged group targeting the pYbinding site leads to particularly active probes, in certainembodiments. For example, the docking study of the 3-phosphomethylcompound 5 (R═CH₂PO₃ ²⁻) showed all of the phosphonate groups of the 20docking poses are tightly clustered together and located in the pYbinding site, indicating strong electrostatic and H-bond interactionswith the residues Arg609 and Lys591 (FIG. 18).

EXP IC. Modifications 3 and 4. Collectively, Modifications 3 and 4 testthe effects of changing the substituents at the 4, 5, and 6-positions.The —OH at 4-position may be superior to —OR, in certain aspects. Onecan test whether the H atom in —OH is responsible for a better activityby synthesizing compounds 6 (acylated or alkylated 5), as schematicallyshown below. In addition, dehydroxy compounds 7 may also be made,starting from 3-bromonaphthyl-1-amine.

Regarding the general synthetic methods for modifying positions 5 and 6,one can first synthesize about a dozen of these compounds in thiscategory and if very active compounds emerge, one can make morecompounds to optimize the activity for these two positions.

EXP ID. Modification 5. The only two compounds not included in the SARanalysis (due to a different 4-substituent) are shown here, as well astheir inhibitory activities against Stat3:

Despite the weak activity, masking the polar H of the sulfamide for thesecond compound is favorable, in certain aspects, which provides an easyroute to making more potent probes. One can therefore use the followingmethod to make a series of N-acyl or N-alkyl sulfamides 5:

Example 9 Identification of Stat3-Selective Chemical Probes fromSulfamide Compounds Synthesized in Example 11

Each novel sulfamide compound is tested for the ability to inhibit Stat3binding to its phosphopeptide ligand by SPR and the ability to blockIL-6-stimulated cytoplasmic-to-nuclear translocation in the HTFM assay.Probes with activity in these assays equivalent to or greater than themost active 2nd generation compounds are tested for inhibition ofIL-6-stimulated Stat3 phosphorylation and lack of ability to inhibitIFN-γ-stimulated Stat1 phosphorylation as outlined below.

EXP IIA. Stat3/pY-peptide SPR binding inhibition assay. Stat3 pY-peptidebinding assays is performed at 25° C. using a BIAcore 3000 biosensor asdescribed (Xu et al., 2009). Briefly, phosphorylated and controlnonphosphorylated biotinylated EGFR derived dodecapeptides based on thesequence surrounding Y1068 are immobilized on a streptavidin coatedsensor chip (BIAcore Inc., Piscataway N.J.). The binding of Stat3 isperformed in 20 mM Tris buffer pH 8 containing 2 mM β-mercaptoethanol ata flow rate of 10 uL/min for 1-2 minute. Aliquots of Stat3 at 500 nM arepremixed with compound to achieve a final concentration of 1-1,000 uMand incubated at 4° C. prior to being injected onto the sensor chip. Thechip is regenerated by injecting 10 uL of 100 mM glycine at pH 1.5 aftereach sample injection. A control (Stat3 with DMSO but without compound)is run at the beginning and the end of each cycle (40 sample injections)to ensure that the integrity of the sensor chip is maintained throughoutthe cycle run. The average of the two controls is normalized to 100% andused to evaluate the effect of each compound on Stat3 binding. Responsesare normalized by dividing the value at 2 min by the response obtainedin the absence of compounds at 2 min and multiplying by 100. IC₅₀ valuesare determined by plotting % maximum response as a function of logconcentration of compound and fitting the experimental points to acompetitive binding model using a four parameter logistic equation:R=R_(high)−(R_(high)−R_(low))/(1+conc/A1)^(A2), where R=percent responseat inhibitor concentration, R_(high)=percent response with no compound,R_(low)=percent response at highest compound concentration, A2=fittingparameter (slope) and A1=IC₅₀ (BIAevaluation Software version 4.1).

EXP IIB. High throughput fluorescence microscopy (HTFM),cytoplasm-to-nucleus translocation inhibition assays. HTFM ofMEF/GFP-Stat3α cells is performed to assess the ability of probes toinhibit GFP-Stat3 cytoplasmic-to-nuclear translocation, as described (Xuet al., 2009), using the robotic system available as part of the John S.Dunn Gulf Coast Consortium for Chemical Genomics at the University ofTexas-Houston School of Medicine. Briefly, cells are seeded into 96-wellCC3 plates at a density of 5,000 cells/well and cultured under standardconditions until 85-90% confluent. Cells are pre-treated with compoundfor 1 hour at 37° C. then stimulated with IL-6 (100 ng/ml) and IL-6sR(150 ng/ml) for 30 minutes. Cells are fixed with 4% formaldehyde in PEMBuffer (80 mM Potassium PIPES, pH 6.8, 5 mM EGTA pH 7.0, 2 mM MgCl₂) for30 minutes at 4° C., quenched in 1 mg/ml of NaBH₄ (Sigma) in PEM bufferand counterstained for 1 min in 4,6-diamidino-2-phenylindole (DAPI;Sigma; 1 mg/ml) in PEM buffer. Plates are analyzed by automated HTFMusing the Cell Lab IC Image Cytometer (IC100) platform andCytoshopVersion 2.1 analysis software (Beckman Coulter).

Nuclear translocation is quantified by using the fraction localized inthe nucleus (FLIN) measurement. FLIN values are normalized bysubtracting the FLIN for unstimulated cells then dividing thisdifference by the maximum difference (delta, A) in FLIN (FLIN in cellsstimulated with IL-6/sIL-6R in the absence of compound minus FLIN ofunstimulated cells). This ratio is multiplied by 100 to obtain thepercentage of maximum difference in FLIN and is plotted as a function ofthe log compound concentration. The best-fitting curve and IC₅₀ valueare determined using 4-Parameter LogisticModel/Dose Response/XLfit 4.2,IDBS software.

EXP IIC. Ligand-mediated pStat3 and pStat1 inhibition assays. Newlysynthesized Stat3 probes with activity equivalent to or greater thanparent compound 188 in the SPR and HTFM assays will be tested for theability to selectively inhibit ligand-mediated phosphorylation of Stat3as described (Xu et al., 2009). Briefly, human hepatocellular carcinomacells (HepG2) are grown in 6-well plates and pretreated with compounds(0, 0.1, 0.3, 1, 3, 10, 30, 100 μM) for 1 hour then stimulated underoptimal conditions with either interleukin-6 (IL-6; 30 ng/ml for 30 min)to activate Stat3 or interferon gamma (IFN-γ; 30 ng/ml for 30 min) toactivate Stat1. Cells are harvested and proteins extracted usinghigh-salt buffer, mixed with 2× sodium dodecyl sulfate (SDS) samplebuffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20% glycerol;10%2-mercaptoethanol) at a 1:1 ratio then heated for 5 minutes at 100°C. Proteins (20 μg) are separated by 7.5% SDS-PAGE and transferred topolyvinylidene fluoride (PVDF) membrane (Millipore, Waltham, Mass.) andimmunoblotted. Membranes are probed serially with antibody against Stat1pY701 or Stat3 pY705 followed by antibody against Stat1 or Stat3(Transduction labs, Lexington, Ky.) then antibody against β-actin(Abcam, Cambridge, Mass.). Membranes are stripped between antibodyprobings using Restore™ Western Blot Stripping Buffer (Thermo FisherScientific Inc., Waltham, Mass.) per the manufacturer's instructions.Horseradish peroxidase-conjugated goat-anti-mouse IgG is used as thesecondary antibody (Invitrogen Carlsbad, Calif.) and the membranes aredeveloped with enhanced chemiluminescence (ECL) detection system(Amersham Life Sciences Inc.; Arlington Heights, Ill.). Band intensitiesare quantified by densitometry. The value of each pStat3 band is dividedby its corresponding total Stat3 band intensity; the results arenormalized to the DMSO-treated control value. This value was plotted asa function of the log compound concentration. The best-fitting curve isdetermined using 4-Parameter Logistic Model/Dose Response/XLfit 4.2,IDBS software and was used to calculate the IC₅₀ value.

EXP IID. Molecular modeling of probe-Stat3 interactions. The results ofmodeling of the binding of the first generation probe to the Stat3 vs.Stat1 SH2 domains suggested that the basis for experimental selectivityof probes for Stat3 vs. Stat1 rested on the ability of the probes tohave greater interaction with the hydrophobic binding site within thepY-peptide binding pocket of Stat3 compared to Stat1. Thus, thehydrophobic binding site served as a selectivity filter. To test if thisremains the case for newly synthesized 3rd generation probes, one canuse 2 complementary docking programs GLIDE (Schrödinger) and ICM(MolSoft) to determine the lowest energy docking configuration of eachprobe within the pY-peptide binding domain of Stat3 and Stat1 SH2domain. One can review the computational models of each probe in acomplex with the Stat3 vs. Stat1 SH2 domain and, in particular, comparethe van der Waals energies and determine if they are equivalent fortheir interaction with the Stat3 SH2 domain vs. the Stat1 SH2 domain. Itwas this calculation that determined the selectivity of 1st generationprobes for Stat3 vs. Stat1. In particular, van der Waals energycalculations implicated residues that form the hydrophobic binding site(W623, Q635, V637, Y640 and Y657) as critical for this selectivity.

In specific embodiments of the invention, there is identification ofprobes with one log or greater activity than 2^(nd) generation probes inSPR, HTFM and pStat3 assays. Furthermore, in certain aspects some of themost active 3^(rd) generation probes that emerge from this analysis areselective for Stat3 vs. Stat1 based on their greater interaction withthe hydrophobic binding site within the Stat3 vs. Stat1 SH2 pY-peptidebinding pocket.

Example 10 Exemplary Compositions of the Disclosure

Exemplary composition(s) of the disclosure are provided in Tables 6-11below.

TABLE 6 IDNUMBER Structure Formula structure MW LogP F1566-0306

C22H17NO3S2 407.5137 5.846 F1566-0318

C23H19NO3S2 421.5408 6.144 F1566-0330

C22H16ClNO3S2 441.9587 6.438 F1566-0342

C22H16BrNO3S2 486.4097 6.644 F1566-0366

C24H21NO3S2 435.5679 6.477 F1566-0414

C24H21NO3S2 435.5679 6.477 F1566-0438

C24H21NO3S2 435.5679 6.619 F1566-0450

C23H19NO4S2 437.5402 5.802 F1566-0462

C24H21NO4S2 451.5673 6.143 F1566-0486

C26H25NO3S2 463.6221 7.345 F1566-0510

C26H19NO3S2 457.5742 7.105 F1566-0546

C22H16N2O5S2 452.5112 5.818 F1566-0558

C23H18N2O5S2 466.5383 6.114 F1566-0618

C20H15NO3S3 413.5395 5.359 F1566-1606

C25H18N2O3S2 458.5618 6.046 F1566-1818

C18H17NO3S2 359.4691 4.705 F1566-1832

C19H19NO3S2 373.4962 5.147 F1566-1846

C20H21NO3S2 387.5233 5.589 F1566-1860

C17H15NO3S2 345.442 4.192 F5749-0371

C22H16N2O5S2 452.5112 5.781 F5749-0372

C22H23NO3S2 413.5615 6.171 F5749-0373

C25H23NO4S2 465.5944 6.468 F5749-0374

C23H18ClNO4S2 471.9852 6.429 F5749-0375

C24H21NO3S2 435.5679 6.438 F5749-0376

C24H19NO5S2 465.5507 5.787 F5749-0377

C24H20N2O4S2 464.566 5.137 F5749-0378

C24H21NO5S2 467.5667 5.54474 F5749-0379

C24H19NO5S2 465.5507 5.441 F5749-0380

C21H16N2O3S2 408.5013 4.613 F5749-0381

C18H18N2O3S2 374.4838 3.74 F5749-0382

C24H21NO3S2 435.5679 6.477 F5749-0383

C22H16N2O5S2 452.5112 5.779 F5749-0384

C23H19NO3S2 421.5408 5.98 F5749-0385

C20H14ClNO3S3 447.9845 6.649 F5749-0386

C22H15F2NO3S2 443.4946 6.187 F5749-0387

C21H19N3O3S2 425.5319 4.956 F5749-0388

C21H18N2O4S2 426.5166 4.99 F5749-0389

C23H22N2O5S2 470.5702 3.633 F5749-0390

C23H18FNO4S2 455.5306 5.99 F5749-0391

C24H21NO4S2 451.5673 6.135 F5749-0392

C26H20N2O3S2 472.5889 6.305 F5749-0393

C22H19NO3S3 441.5936 6.497 F5749-0394

C21H17NO3S3 427.5665 6.022 F5749-0395

C24H19NO3S2 433.5519 6.204 F5749-0396

C22H16FNO3S2 425.5041 5.997 F5749-0397

C23H19NO4S2 437.5402 5.839 F5749-0398

C22H16FNO3S2 425.5041 6.036 F5749-0399

C22H15ClFNO3S2 459.9492 6.626 F5749-0400

C23H16F3NO4S2 491.5115 7.24476 F5749-0401

C23H18ClNO3S2 455.9858 6.771 F5749-0402

C24H19NO4S2 449.5513 5.736 F5749-0403

C24H19NO4S2 449.5513 5.699 F5749-0404

C23H18ClNO3S2 455.9858 6.732 F5749-0405

C23H19NO4S2 437.5402 5.8 F5749-0406

C24H21NO4S2 451.5673 6.141 F5749-0407

C22H15F2NO3S2 443.4946 6.148 F5749-0408

C19H19NO3S2 373.4962 5.339 F5749-0409

C23H16F3NO3S2 475.5121 6.81776 F5749-0410

C23H16F3NO3S2 475.5121 6.78076 F5749-0411

C22H16ClNO3S2 441.9587 6.475 F5749-0412

C23H17Cl2NO3S2 490.4308 7.398 F5749-0413

C22H15F2NO3S2 443.4946 6.187 F5749-0414

C25H23NO3S2 449.595 7.061 F5749-0415

C26H23NO3S2 461.6061 6.933 F5749-0416

C26H20N2O5S2 504.5877 4.973 F5749-0417

C27H22N2O5S2 518.6148 5.415 F5749-0418

C23H20N2O4S3 484.6189 5.149 F5749-0419

C20H15N3O5S2 441.4877 2.891 F5749-0420

C25H20N2O4S2 476.5772 5.042 F5749-0421

C24H18N2O4S2 462.5501 4.954 F5749-0422

C22H19N3O5S2 469.5418 2.955 F5749-0423

C26H22N2O4S2 490.6042 5.277 F5749-0424

C23H18FNO3S2 439.5312 6.133 F5749-0425

C23H18FNO3S2 439.5312 6.17 F5749-0426

C25H23NO4S2 465.5944 6.206 F5749-0427

C28H25N3O3S2 515.6578 6.125 F5749-0428

C19H15N3O3S2 397.4777 3.986 F5749-0429

C27H23N3O3S2 501.6307 5.991 F5749-0430

C29H23NO5S2 529.6384 7.16174 F5749-0431

C28H20ClNO4S2 534.0569 8.046 F5749-0432

C29H23NO4S2 513.639 7.754 F5749-0433

C23H15ClF3NO3S2 509.9571 7.40776 F5749-0434

C28H21NO4S2 499.6119 7.456 F5749-0435

C22H16BrNO3S2 486.4097 6.642 F5749-0436

C22H16BrNO3S2 486.4097 6.681 F5749-0437

C22H15BrFNO3S2 504.4002 6.832 F5749-0438

C23H15BrF3NO3S2 554.4081 7.61376 F5749-0439

C22H16ClNO3S2 441.9587 6.436 F5749-0440

C22H17NO5S3 471.5765 5.046 F5749-0441

C23H16F3NO4S2 491.5115 7.24276

TABLE 7 IDNUMBER Structure Formula structure MW LogP F0808-0081

C28H23NO4S 469.5638 7.101 F0808-0084

C28H23NO5S 485.5632 6.767 F0808-0085

C26H18BrNO4S 520.4057 7.268 F0808-0086

C28H23NO4S 469.5638 7.243 F0808-0089

C30H21NO4S 491.5702 7.729 F0808-0091

C26H18FNO4S 459.5001 6.623 F0808-0092

C28H23NO4S 469.5638 7.101 F0808-0094

C26H18ClNO4S 475.9547 7.062 F1269-0222

C24H17NO4S2 447.5354 5.983 F1269-2003

C27H20N2O6S 500.5343 6.738 F1566-1138

C29H20N2O4S 492.5578 6.67 F5749-0001

C21H17NO4S 379.4379 4.816 F5749-0002

C26H18N2O6S 486.5072 6.405 F5749-0003

C26H25NO4S 447.5575 6.795 F5749-0004

C29H25NO5S 499.5903 7.092 F5749-0005

C27H20ClNO5S 505.9812 7.053 F5749-0006

C28H23NO4S 469.5638 7.062 F5749-0007

C28H21NO6S 499.5467 6.411 F5749-0008

C28H22N2O5S 498.5619 5.761 F5749-0009

C28H23NO6S 501.5626 6.16874 F5749-0010

C28H21NO6S 499.5467 6.065 F5749-0011

C25H18N2O4S 442.4972 5.237 F5749-0012

C22H19NO4S 393.465 5.329 F5749-0013

C28H23NO6S 501.5626 6.417 F5749-0014

C22H20N2O4S 408.4797 4.364 F5749-0015

C28H23NO4S 469.5638 7.101 F5749-0016

C26H18N2O6S 486.5072 6.403 F5749-0017

C23H21NO4S 407.4921 5.771 F5749-0018

C27H21NO4S 455.5367 6.604 F5749-0019

C24H23NO4S 421.5192 6.213 F5749-0020

C24H16ClNO4S2 481.9804 7.273 F5749-0021

C26H17F2NO4S 477.4905 6.811 F5749-0022

C25H21N3O4S 459.5278 5.58 F5749-0023

C25H20N2O5S 460.5126 5.614 F5749-0024

C27H24N2O6S 504.5661 4.257 F5749-0025

C27H20FNO5S 489.5266 6.614 F5749-0026

C28H23NO5S 485.5632 6.759 F5749-0027

C30H22N2O4S 506.5848 6.929 F5749-0028

C26H21NO4S2 475.5896 7.121 F5749-0029

C25H19NO4S2 461.5625 6.646 F5749-0030

C28H21NO4S 467.5479 6.828 F5749-0031

C26H18FNO4S 459.5001 6.621 F5749-0032

C27H21NO5S 471.5361 6.463 F5749-0033

C26H18FNO4S 459.5001 6.66 F5749-0034

C26H17ClFNO4S 493.9451 7.25 F5749-0035

C27H18F3NO5S 525.5074 7.86876 F5749-0036

C27H20ClNO4S 489.9818 7.395 F5749-0037

C28H21NO5S 483.5473 6.36 F5749-0038

C28H21NO5S 483.5473 6.323 F5749-0039

C27H20ClNO4S 489.9818 7.356 F5749-0040

C27H21NO5S 471.5361 6.424 F5749-0041

C28H23NO5S 485.5632 6.765 F5749-0042

C26H17F2NO4S 477.4905 6.772 F5749-0043

C23H21NO4S 407.4921 5.963 F5749-0044

C27H18F3NO4S 509.508 7.44176 F5749-0045

C27H18F3NO4S 509.508 7.40476 F5749-0046

C26H18ClNO4S 475.9547 7.099 F5749-0047

C27H19Cl2NO4S 524.4268 8.022 F5749-0048

C26H17F2NO4S 477.4905 6.811 F5749-0049

C29H25NO4S 483.5909 7.685 F5749-0050

C30H25NO4S 495.6021 7.557 F5749-0051

C30H22N2O6S 538.5836 5.597 F5749-0052

C31H24N2O6S 552.6107 6.039 F5749-0053

C27H22N2O5S2 518.6148 5.773 F5749-0054

C24H17N3O6S 475.4836 3.515 F5749-0055

C29H22N2O5S 510.5731 5.666 F5749-0056

C28H20N2O5S 496.546 5.578 F5749-0057

C26H21N3O6S 503.5378 3.579 F5749-0058

C30H24N2O5S 524.6002 5.901 F5749-0059

C27H20FNO4S 473.5272 6.757 F5749-0060

C27H20FNO4S 473.5272 6.794 F5749-0061

C29H25NO5S 499.5903 6.83 F5749-0062

C32H27N3O4S 549.6537 6.749 F5749-0063

C23H17N3O4S 431.4736 4.61 F5749-0064

C31H25N3O4S 535.6266 6.615 F5749-0065

C33H25NO6S 563.6343 7.78574 F5749-0066

C32H22ClNO5S 568.0528 8.67 F5749-0067

C33H25NO5S 547.6349 8.378 F5749-0068

C27H17ClF3NO4S 543.953 8.03176 F5749-0069

C32H23NO5S 533.6078 8.08 F5749-0070

C26H18BrNO4S 520.4057 7.266 F5749-0071

C26H18BrNO4S 520.4057 7.305 F5749-0072

C26H17BrFNO4S 538.3961 7.456 F5749-0073

C27H17BrF3NO4S 588.404 8.23776 F5749-0074

C26H18ClNO4S 475.9547 7.06 F5749-0075

C26H19NO6S2 505.5724 5.67 F5749-0076

C27H18F3NO5S 525.5074 7.86676

TABLE 8 IDNUMBER Structure Formula structure MW LogP F1566-0329

C26H20N2O3S2 472.5889 6.344 F1566-0341

C25H17ClN2O3S2 493.0068 6.638 F1566-0353

C25H17BrN2O3S2 537.4578 6.844 F1566-0377

C27H22N2O3S2 486.616 6.677 F1566-0425

C27H22N2O3S2 486.616 6.677 F1566-0449

C27H22N2O3S2 486.616 6.819 F1566-0473

C27H22N2O4S2 502.6154 6.343 F1566-0497

C29H26N2O3S2 514.6702 7.545 F1566-0521

C29H20N2O3S2 508.6224 7.305 F1566-0557

C25H17N3O5S2 503.5593 6.018 F1566-0569

C26H19N3O5S2 517.5864 6.314 F1566-0617

C27H22N2O5S2 518.6148 5.993 F1566-0629

C23H16N2O3S3 464.5876 5.559 F1566-1608

C28H19N3O3S2 509.6099 6.246 F1566-1821

C21H18N2O3S2 410.5172 4.905 F1566-1835

C22H20N2O3S2 424.5443 5.347 F1566-1849

C23H22N2O3S2 438.5714 5.789 F1566-1863

C20H16N2O3S2 396.4901 4.392 F5749-0077

C25H17N3O5S2 503.5593 5.981 F5749-0078

C25H24N2O3S2 464.6096 6.371 F5749-0079

C28H24N2O4S2 516.6425 6.668 F5749-0080

C26H19ClN2O4S2 523.0333 6.629 F5749-0081

C27H22N2O3S2 486.616 6.638 F5749-0082

C27H20N2O5S2 516.5989 5.987 F5749-0083

C27H21N3O4S2 515.6141 5.337 F5749-0084

C27H22N2O5S2 518.6148 5.74474 F5749-0085

C27H20N2O5S2 516.5989 5.641 F5749-0086

C24H17N3O3S2 459.5494 4.813 F5749-0087

C21H19N3O3S2 425.5319 3.94 F5749-0088

C27H22N2O3S2 486.616 6.677 F5749-0089

C25H17N3O5S2 503.5593 5.979 F5749-0090

C26H20N2O3S2 472.5889 6.18 F5749-0091

C23H15ClN2O3S3 499.0326 6.849 F5749-0092

C25H16F2N2O3S2 494.5427 6.387 F5749-0093

C24H20N4O3S2 476.58 5.156 F5749-0094

C24H19N3O4S2 477.5647 5.19 F5749-0095

C26H23N3O5S2 521.6183 3.833 F5749-0096

C26H19FN2O4S2 506.5787 6.19 F5749-0097

C27H22N2O4S2 502.6154 6.335 F5749-0098

C29H21N3O3S2 523.637 6.505 F5749-0099

C25H20N2O3S3 492.6418 6.697 F5749-0100

C24H18N2O3S3 478.6147 6.222 F5749-0101

C27H20N2O3S2 484.6001 6.404 F5749-0102

C25H17FN2O3S2 476.5522 6.197 F5749-0103

C26H20N2O4S2 488.5883 6.039 F5749-0104

C25H17FN2O3S2 476.5522 6.236 F5749-0105

C25H16ClFN2O3S2 510.9973 6.826 F5749-0106

C26H17F3N2O4S2 542.5596 7.44476 F5749-0107

C26H19ClN2O3S2 507.0339 6.971 F5749-0108

C27H20N2O4S2 500.5995 5.936 F5749-0109

C27H20N2O4S2 500.5995 5.899 F5749-0110

C26H19ClN2O3S2 507.0339 6.932 F5749-0111

C26H20N2O4S2 488.5883 6 F5749-0112

C27H22N2O4S2 502.6154 6.341 F5749-0113

C25H16F2N2O3S2 494.5427 6.348 F5749-0114

C22H20N2O3S2 424.5443 5.539 F5749-0115

C26H17F3N2O3S2 526.5602 7.01776 F5749-0116

C26H17F3N2O3S2 526.5602 6.98076 F5749-0117

C25H17ClN2O3S2 493.0068 6.675 F5749-0118

C26H18Cl2N2O3S2 541.479 7.598 F5749-0119

C25H16F2N2O3S2 494.5427 6.387 F5749-0120

C28H24N2O3S2 500.6431 7.261 F5749-0121

C29H24N2O3S2 512.6542 7.133 F5749-0122

C29H21N3O5S2 555.6358 5.173 F5749-0123

C30H23N3O5S2 569.6629 5.615 F5749-0124

C26H21N3O4S3 535.667 5.349 F5749-0125

C23H16N4O5S2 492.5358 3.091 F5749-0126

C28H21N3O4S2 527.6253 5.242 F5749-0127

C27H19N3O4S2 513.5982 5.154 F5749-0128

C25H20N4O5S2 520.59 3.155 F5749-0129

C29H23N3O4S2 541.6524 5.477 F5749-0130

C26H19FN2O3S2 490.5793 6.333 F5749-0131

C26H19FN2O3S2 490.5793 6.37 F5749-0132

C28H24N2O4S2 516.6425 6.406 F5749-0133

C31H26N4O3S2 566.7059 6.325 F5749-0134

C22H16N4O3S2 448.5258 4.186 F5749-0135

C30H24N4O3S2 552.6788 6.191 F5749-0136

C32H24N2O5S2 580.6865 7.36174 F5749-0137

C31H21ClN2O4S2 585.105 8.246 F5749-0138

C32H24N2O4S2 564.6871 7.954 F5749-0139

C26H16ClF3N2O3S2 561.0052 7.60776 F5749-0140

C31H22N2O4S2 550.66 7.656 F5749-0141

C25H17BrN2O3S2 537.4578 6.842 F5749-0142

C25H17BrN2O3S2 537.4578 6.881 F5749-0143

C25H16BrFN2O3S2 555.4483 7.032 F5749-0144

C26H16BrF3N2O3S2 605.4562 7.81376 F5749-0145

C25H17ClN2O3S2 493.0068 6.636 F5749-0146

C25H18N2O5S3 522.6246 5.246 F5749-0147

C26H17F3N2O4S2 542.5596 7.44276

TABLE 9 ID NUMBER Structure Formula structure MW Log P F1565-0253

C18H14N4O3S2 398.4653 3.698 F1566-0328

C19H16N4O3S2 412.4924 3.996 F1566-0340

C18H13ClN4O3S2 432.9103 4.29 F1566-0520

C22H16N4O3S2 448.5258 4.957 F1566-0556

C18H13N5O5S2 443.4628 3.67 F1566-0568

C19H15N5O5S2 457.4899 3.966 F1566-0616

C20H18N4O5S2 458.5183 3.645 F1566-0628

C16H12N4O3S3 404.491 3.211 F5749-0148

C13H12N4O3S2 336.3936 2.044 F5749-0149

C18H13N5O5S2 443.4628 3.633 F5749-0150

C18H20N4O3S2 404.5131 4.023 F5749-0151

C21H20N4O4S2 456.546 4.32 F5749-0152

C19H15ClN4O4S2 462.9368 4.281 F5749-0153

C20H18N4O3S2 426.5195 4.29 F5749-0154

C20H16N4O5S2 456.5023 3.639 F5749-0155

C20H17N5O4S2 455.5176 2.989 F5749-0156

C20H18N4O5S2 458.5183 3.39674 F5749-0157

C20H16N4O5S2 456.5023 3.293 F5749-0158

C17H13N5O3S2 399.4529 2.465 F5749-0159

C14H14N4O3S2 350.4207 2.557 F5749-0160

C14H15N5O3S2 365.4354 1.592 F5749-0161

C20H18N4O3S2 426.5195 4.329 F5749-0162

C18H13N5O5S2 443.4628 3.631 F5749-0163

C15H16N4O3S2 364.4478 2.999 F5749-0164

C19H16N4O3S2 412.4924 3.832 F5749-0165

C16H18N4O3S2 378.4749 3.441 F5749-0166

C16H11ClN4O3S3 438.9361 4.501 F5749-0167

C18H12F2N4O3S2 434.4461 4.039 F5749-0168

C17H16N6O3S2 416.4835 2.808 F5749-0169

C17H15N5O4S2 417.4682 2.842 F5749-0170

C19H19N5O5S2 461.5218 1.485 F5749-0171

C19H15FN4O4S2 446.4822 3.842 F5749-0172

C20H18N4O4S2 442.5189 3.987 F5749-0173

C22H17N5O3S2 463.5405 4.157 F5749-0174

C21H15N5O3S2 449.5134 3.898 F5749-0175

C18H16N4O3S3 432.5452 4.349 F5749-0176

C17H14N4O3S3 418.5181 3.874 F5749-0177

C20H16N4O3S2 424.5035 4.056 F5749-0178

C18H13FN4O3S2 416.4557 3.849 F5749-0179

C19H16N4O4S2 428.4918 3.691 F5749-0180

C18H13FN4O3S2 416.4557 3.888 F5749-0181

C18H12ClFN4O3S2 450.9007 4.478 F5749-0182

C19H13F3N4O4S2 482.4631 5.09676 F5749-0183

C19H15ClN4O3S2 446.9374 4.623 F5749-0184

C20H16N4O4S2 440.5029 3.588 F5749-0185

C20H16N4O4S2 440.5029 3.551 F5749-0186

C19H15ClN4O3S2 446.9374 4.584 F5749-0187

C19H16N4O4S2 428.4918 3.652 F5749-0188

C20H18N4O4S2 442.5189 3.993 F5749-0189

C18H12F2N4O3S2 434.4461 4 F5749-0190

C15H16N4O3S2 364.4478 3.191 F5749-0191

C19H13F3N4O3S2 466.4637 4.66976 F5749-0192

C19H13F3N4O3S2 466.4637 4.63276 F5749-0193

C18H13ClN4O3S2 432.9103 4.327 F5749-0194

C19H14Cl2N4O3S2 481.3824 5.25 F5749-0195

C18H12F2N4O3S2 434.4461 4.039 F5749-0196

C21H20N4O3S2 440.5466 4.913 F5749-0197

C22H20N4O3S2 452.5577 4.785 F5749-0198

C22H17N5O5S2 495.5393 2.825 F5749-0199

C23H19N5O5S2 509.5664 3.267 F5749-0200

C19H17N5O4S3 475.5704 3.001 F5749-0201

C16H12N6O5S2 432.4392 0.743 F5749-0202

C21H17N5O4S2 467.5287 2.894 F5749-0203

C20H15N5O4S2 453.5017 2.806 F5749-0204

C18H16N6O5S2 460.4934 0.807 F5749-0205

C22H19N5O4S2 481.5558 3.129 F5749-0206

C19H15FN4O3S2 430.4828 3.985 F5749-0207

C19H15FN4O3S2 430.4828 4.022 F5749-0208

C21H20N4O4S2 456.546 4.058 F5749-0209

C24H22N6O3S2 506.6093 3.977 F5749-0210

C15H12N6O3S2 388.4293 1.838 F5749-0211

C23H20N6O3S2 492.5823 3.843 F5749-0212

C25H20N4O5S2 520.59 5.01374 F5749-0213

C24H17ClN4O4S2 525.0085 5.898 F5749-0214

C25H20N4O4S2 504.5906 5.606 F5749-0215

C19H12ClF3N4O3S2 500.9087 5.25976 F5749-0216

C24H18N4O4S2 490.5635 5.308 F5749-0217

C18H13BrN4O3S2 477.3613 4.494 F5749-0218

C18H13BrN4O3S2 477.3613 4.533 F5749-0219

C18H12BrFN4O3S2 495.3517 4.684 F5749-0220

C19H12BrF3N4O3S2 545.3597 5.46576 F5749-0221

C18H13ClN4O3S2 432.9103 4.288 F5749-0222

C18H14N4O5S3 462.5281 2.898 F5749-0223

C19H13F3N4O4S2 482.4631 5.09476

TABLE 10 ID NUMBER Structure Formula structure MW LogP F0808-0128

C25H20N2O353 492.6418 6.892 F0808-0132

C23H16N2O3S3 464.5876 6.261 F0808-0133

C23H15ClN2O3S3 499.0326 6.853 F0808-0134

C24H18N2O3S3 478.6147 6.559 F0808-0136

C25H20N2O3S3 492.6418 7.034 F0808-0137

C23H15BrN2O3S3 543.4836 7.059 F1269-0225

C21H14N2O3S4 470.6133 5.774 F1269-1420

C24H18N2O4S3 494.6141 6.217 F1566-1144

C26H17N3O3S3 515.6357 6.461 F1566-1584

C24H17N3O5S3 523.6122 6.529 F1566-1596

C25H20N2O5S3 524.6406 6.208 F1566-1816

C19H16N2O3S3 416.543 5.12 F1566-1830

C20H18N2O3S3 430.5701 5.562 F1566-1844

C21H20N2O3S3 444.5972 6.004 F1566-1858

C18H14N2O3S3 402.5159 4.607 F5749-0224

C23H15N3O5S3 509.5851 6.196 F5749-0225

C23H22N2O3S3 470.6354 6.586 F5749-0226

C26H22N2O4S3 522.6682 6.883 F5749-0227

C24H17ClN2O4S3 529.0591 6.844 F5749-0228

C25H20N2O3S3 492.6418 6.853 F5749-0229

C25H18N2O5S3 522.6246 6.202 F5749-0230

C25H19N3O4S3 521.6399 5.552 F5749-0231

C25H20N2O5S3 524.6406 5.95974 F5749-0232

C25H18N2O5S3 522.6246 5.856 F5749-0233

C22H15N3O3S3 465.5752 5.028 F5749-0234

C19H17N3O3S3 431.5576 4.155 F5749-0235

C25H20N2O3S3 492.6418 6.892 F5749-0236

C23H15N3O5S3 509.5851 6.194 F5749-0237

C24H18N2O3S3 478.6147 6.395 F5749-0238

C21H13ClN2O3S4 505.0584 7.064 F5749-0239

C23H14F2N2O3S3 500.5684 6.602 F5749-0240

C22H18N4O3S3 482.6058 5.371 F5749-0241

C22H17N3O4S3 483.5905 5.405 F5749-0242

C24H21N3O5S3 527.6441 4.048 F5749-0243

C24H17FN2O4S3 512.6045 6.405 F5749-0244

C25H20N2O4S3 508.6412 6.55 F5749-0245

C27H19N3O3S3 529.6628 6.72 F5749-0246

C23H18N2O3S4 498.6675 6.912 F5749-0247

C22H16N2O3S4 484.6404 6.437 F5749-0248

C25H18N2O3S3 490.6258 6.619 F5749-0249

C23H15FN2O3S3 482.578 6.412 F5749-0250

C24H18N2O4S3 494.6141 6.254 F5749-0251

C23H15FN2O3S3 482.578 6.451 F5749-0252

C23H14ClFN2O3S3 517.023 7.041 F5749-0253

C24H15F3N2O4S3 548.5854 7.65976 F5749-0254

C24H17ClN2O3S3 513.0597 7.186 F5749-0255

C25H18N2O4S3 506.6252 6.151 F5749-0256

C25H18N2O4S3 506.6252 6.114 F5749-0257

C24H17ClN2O3S3 513.0597 7.147 F5749-0258

C24H18N2O4S3 494.6141 6.215 F5749-0259

C25H20N2O4S3 508.6412 6.556 F5749-0260

C23H14F2N2O3S3 500.5684 6.563 F5749-0261

C20H18N2O3S3 430.5701 5.754 F5749-0262

C24H15F3N2O3S3 532.586 7.23276 F5749-0263

C24H15F3N2O3S3 532.586 7.19576 F5749-0264

C23H15ClN2O3S3 499.0326 6.89 F5749-0265

C24H16Cl2N2O3S3 547.5047 7.813 F5749-0266

C23H14F2N2O3S3 500.5684 6.602 F5749-0267

C26H22N2O3S3 506.6688 7.476 F5749-0268

C27H22N2O3S3 518.68 7.348 F5749-0269

C27H19N3O5S3 561.6616 5.388 F5749-0270

C28H21N3O5S3 575.6887 5.83 F5749-0271

C24H19N3O4S4 541.6927 5.564 F5749-0272

C21H14N4O5S3 498.5615 3.306 F5749-0273

C26H19N3O4S3 533.651 5.457 F5749-0274

C25H17N3O4S3 519.6239 5.369 F5749-0275

C23H18N4O5S3 526.6157 3.37 F5749-0276

C27H21N3O4S3 547.6781 5.692 F5749-0277

C24H17FN2O3S3 496.6051 6.548 F5749-0278

C24H17FN2O3S3 496.6051 6.585 F5749-0279

C26H22N2O4S3 522.6682 6.621 F5749-0280

C29H24N4O3S3 572.7316 6.54 F5749-0281

C20H14N4O3S3 454.5516 4.401 F5749-0282

C28H22N4O3S3 558.7045 6.406 F5749-0283

C30H22N2O5S3 586.7122 7.57674 F5749-0284

C29H19ClN2O4S3 591.1308 8.461 F5749-0285

C30H22N2O4S3 570.7128 8.169 F5749-0286

C24H14ClF3N2O3S3 567.031 7.82276 F5749-0287

C29H20N2O4S3 556.6858 7.871 F5749-0288

C23H15BrN2O3S3 543.4836 7.057 F5749-0289

C23H15BrN2O3S3 543.4836 7.096 F5749-0290

C23H14BrFN2O3S3 561.474 7.247 F5749-0291

C24H14BrF3N2O3S3 611.482 8.02876 F5749-0292

C23H15ClN2O3S3 499.0326 6.851 F5749-0293

C23H16N2O5S4 528.6504 5.461 F5749-0294

C24H15F3N2O4S3 548.5854 7.65776

TABLE 11 ID NUMBER Structure Formula structure MW Log P F0433-0038

C16H12ClNO3S 333.7959 4.192 F0433-0041

C17H14ClNO3S 347.823 4.49 F0433-0044

C16H11Cl2NO3S 368.241 4.784 F0433-0047

C17H14ClNO4S 363.8224 4.148 F0433-0050

C20H14ClNO3S 383.8565 5.451 F0808-1895

C18H16ClNO3S 361.8501 4.823 F0808-1902

C16H11BrClNO3S 412.692 4.99 F0808-1909

C16H11ClN2O5S 378.7935 4.164 F0808-1913

C18H16ClNO3S 361.8501 4.823 F0808-1914

C20H20ClNO3S 389.9043 5.691 F1269-0272

C14H10ClNO3S2 339.8217 3.705 F1269-1995

C17H13ClN2O5S 392.8206 4.46 F1566-1223

C19H13ClN2O3S 384.8441 4.392 F5749-0295

C11H10ClNO3S 271.7243 2.538 F5749-0296

C16H11ClN2O5S 378.7935 4.127 F5749-0297

C16H18ClNO3S 339.8438 4.517 F5749-0298

C19H18ClNO4S 391.8766 4.814 F5749-0299

C17H13Cl2NO4S 398.2675 4.775 F5749-0300

C18H16ClNO3S 361.8501 4.784 F5749-0301

C18H14ClNO5S 391.833 4.133 F5749-0302

C18H15ClN2O4S 390.8483 3.483 F5749-0303

C18H16ClNO5S 393.8489 3.89074 F5749-0304

C18H14ClNO5S 391.833 3.787 F5749-0305

C15H11ClN2O3S 334.7835 2.959 F5749-0306

C12H12ClNO3S 285.7513 3.051 F5749-0307

C18H16ClNO5S 393.8489 4.139 F5749-0308

C12H13ClN2O3S 300.766 2.086 F5749-0309

C18H16ClNO3S 361.8501 4.823 F5749-0310

C16H11ClN2O5S 378.7935 4.125 F5749-0311

C13H14ClNO3S 299.7784 3.493 F5749-0312

C17H14ClNO3S 347.823 4.326 F5749-0313

C14H16ClNO3S 313.8055 3.935 F5749-0314

C14H9Cl2NO3S2 374.2667 4.995 F5749-0315

C16H10ClF2NO3S 369.7768 4.533 F5749-0316

C15H14ClN3O3S 351.8141 3.302 F5749-0317

C15H13ClN2O4S 352.7989 3.336 F5749-0318

C17H17ClN2O5S 396.8524 1.979 F5749-0319

C17H13ClFNO4S 381.8129 4.336 F5749-0320

C18H16ClNO4S 377.8495 4.481 F5749-0321

C20H15ClN2O3S 398.8712 4.651 F5749-0322

C16H14ClNO3S2 367.8759 4.843 F5749-0323

C15H12ClNO3S2 353.8488 4.368 F5749-0324

C18H14ClNO3S 359.8342 4.55 F5749-0325

C16H11ClFNO3S 351.7864 4.343 F5749-0326

C17H14ClNO4S 363.8224 4.185 F5749-0327

C16H11ClFNO3S 351.7864 4.382 F5749-0328

C16H10Cl2FNO3S 386.2314 4.972 F5749-0329

C17H11ClF3NO4S 417.7937 5.59076 F5749-0330

C17H13Cl2NO3S 382.2681 5.117 F5749-0331

C18H14ClNO4S 375.8336 4.082 F5749-0332

C18H14ClNO4S 375.8336 4.045 F5749-0333

C17H13Cl2NO3S 382.2681 5.078 F5749-0334

C17H14ClNO4S 363.8224 4.146 F5749-0335

C18H16ClNO4S 377.8495 4.487 F5749-0336

C16H10ClF2NO3S 369.7768 4.494 F5749-0337

C13H14ClNO3S 299.7784 3.685 F5749-0338

C17H11ClF3NO3S 401.7943 5.16376 F5749-0339

C17H11ClF3NO3S 401.7943 5.12676 F5749-0340

C16H11Cl2NO3S 368.241 4.821 F5749-0341

C17H12Cl3NO3S 416.7131 5.744 F5749-0342

C16H10ClF2NO3S 369.7768 4.533 F5749-0343

C19H18ClNO3S 375.8772 5.407 F5749-0344

C20H18ClNO3S 387.8884 5.279 F5749-0345

C20H15ClN2O5S 430.87 3.319 F5749-0346

C21H17ClN2O5S 444.897 3.761 F5749-0347

C17H15ClN2O4S2 410.9011 3.495 F5749-0348

C14H10ClN3O5S 367.7699 1.237 F5749-0349

C19H15ClN2O4S 402.8594 3.388 F5749-0350

C18H13ClN2O4S 388.8323 3.3 F5749-0351

C16H14ClN3O5S 395.8241 1.301 F5749-0352

C20H17ClN2O4S 416.8865 3.623 F5749-0353

C17H13ClFNO3S 365.8135 4.479 F5749-0354

C17H13ClFNO3S 365.8135 4.516 F5749-0355

C19H18ClNO4S 391.8766 4.552 F5749-0356

C22H20ClN3O3S 441.94 4.471 F5749-0357

C13H10ClN3O3S 323.76 2.332 F5749-0358

C21H18ClN3O3S 427.9129 4.337 F5749-0359

C23H18ClNO5S 455.9206 5.50774 F5749-0360

C22H15Cl2NO4S 460.3392 6.392 F5749-0361

C23H18ClNO4S 439.9212 6.1 F5749-0362

C17H10Cl2F3NO3S 436.2394 5.75376 F5749-0363

C22H16ClNO4S 425.8941 5.802 F5749-0364

C16H11BrClNO3S 412.692 4.988 F5749-0365

C16H11BrClNO3S 412.692 5.027 F5749-0366

C16H10BrClFNO3S 430.6824 5.178 F5749-0367

C17H10BrClF3NO3S 480.6904 5.95976 F5749-0368

C16H11Cl2NO3S 368.241 4.782 F5749-0369

C16H12ClNO5S2 397.8587 3.392 F5749-0370

C17H11ClF3NO4S 417.7937 5.58876

Example 11 Stat3 Activation Initiates a C/EBPδ to Myostatin Pathway thatStimulates Loss of Muscle Mass

As addressed herein, catabolic conditions like chronic kidney disease(CKD) cause loss of muscle mass by unclear mechanisms. In musclebiopsies from CKD patients, activated Stat3 (p-Stat3) was found and itwas considered that p-Stat3 initiates muscle wasting. Mice weregenerated with muscle-specific knockout (KO) that prevents activation ofStat3. In these mice, losses of body and muscle weights were suppressedin models of CKD or acute diabetes. A small molecule that inhibits Stat3activation, produced similar responses suggesting a potential fortranslation strategies. Using C/EBPδ KO mice and C2Cl2 myotubes withknockdown of C/EBPδ or myostatin, it was determined that p-Stat3initiates muscle wasting via C/EBPδ, stimulating myostatin, a negativemuscle growth regulator. C/EBPδ KO also improved survival of CKD mice.It was verified that p-Stat3, C/EBPδ and myostatin were increased inmuscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatinand muscle wasting provides a route for therapeutic targets that preventmuscle wasting.

Muscle Biopsies of Patients with CKD Reveal Inflammation and Stat3Activation

To address the mechanisms underlying muscle wasting, 18 CKD patientsscheduled for peritoneal dialysis catheter insertion and a control groupof 16 age- and gender-matched healthy subjects were studied. Allsubjects led a sedentary lifestyle. In the 18 CKD patients, the BUN andserum creatinine were increased 4- and ˜8-fold respectively over controlsubjects (Table 12).

TABLE 12 Clinical characteristics of patients with chronic kidneydisease (CKD) and controls Controls CKD patients p-value Number ofsubjects 16 18 Age (years) 63 (46-77) 67 (36-79)  0.15 Diabetes 0/16 4/18 Hypertension 0/16 17/18 Atherosclerosis 0/16 14/18 Gender (M:F)13:3 11:7 BMI (kg/m²)  25.4 ± 0.5  27.4 ± 1.2  0.07 BUN (mg/dl)  20.6 ±1.3  89.8 ± 4.6 <0.05 SCr (mg/dL)  0.96 ± 0.04  7.6 ± 1.8 <0.05 eGFR(ml/rnin/1.73 m2)  76.7 ± 3.7  9.1 ± 0.8 <0.05 CSA 0.1 m2) 1873(1100-3389) 1003 (717-1601) <0.003 CRP (mg/dl)  3.21 ± 0.22 10.46 ± 2.98<0.05 Fibrinogen (mg/dl) 291.7 ± 31.7   579 ± 37.5 <0.005

All CKD patients experienced unintentional weight loss in the 3 monthsbefore muscle biopsies were obtained. In CKD patients, the meanestimated protein and calorie intakes were 0.9 g/Kg and 28 Kcal/Kgrespectively, compared to ˜1 g/Kg and 30-32 Kcal/Kg respectively, incontrol healthy subjects (from diet diaries). Even though these intakesof protein and calorie exceed the recommended daily allowance (RDA), 13of the 18 patients were malnourished signified by a the subjectiveglobal assessment level of >2, and serum albumin was low in 11 patients(<3.8 g/100 ml) (Fouque et al., 2008). Even though the body mass indexwas low (<23 Kg/m²) in only 4 subjects, all patients had evidence ofprotein losses: there was a marked reduction in muscle-fibercross-sectional area (CSA) (CKD patients median=1003 μm², range717-1601; controls median=1873 μm², range 1100-3389; p<0.003Mann-Whitney). The fat free mass (FFM) from skin fold thickness (Avesaniet al., 2004) was calculated. Over 3 months, the FFM in CKD patientsdeclined from 45.9±2 to 44.1±2 kg (p<0.05). Regarding drugs that mightinfluence muscle metabolism, no patient was receiving steroids but 14patients were treated with statins; these patients did not have signs ofmyopathy. Characteristics of the CKD patients and control subjects areshown in Table 12. All patients were treated with diuretic (furosemide)at different dosages, lisinopril or doxazosin (17 patients), proton pumpinhibitors (14 patients), platelet aggregation inhibitors (14 patients),insulin therapy (4 patients), oral anticoagulant therapy (1 patient) anderythropoietin (10 patients). Diabetes was well controlled withhemoglobin A1c values <6.5% and fasting plasma glucose levels <110mg/dL. There were increased levels of inflammatory markers in CKDpatients, including circulating C-reactive protein (control; 3.21±0.22vs. CKD; 10.46±2.98 mg/dL; p<0.05) and fibrinogen (control; 291±31.7 vs.CKD; 579±37.5 mg/dL; p<0.005) (Table 12). There also were increasedlevels of IL-6 and TNFα in muscle biopsies compared to results fromcontrol subjects (FIG. 19A). Finally, TNFα mRNA was increased (FIG. 26)and as noted previously, so was IL-6 mRNA (Verzola et al., 2011).

Activated Stat3 protein was significantly increased in muscles of CKDpatients vs. healthy subjects (FIG. 19B). p-Stat3 was principallylocated in nuclei of biopsies as ˜40% of nuclei in muscle biopsies ofCKD patients were positive for p-Stat3 vs. ˜20% in healthy subjects(FIG. 19C). Thus, significant increases in the expressions ofinflammatory cytokines, IL-6 and TNFα, were associated with Stat3activation in muscles of CKD patients who expressed evidence of musclewasting.

Muscle-Specific Stat3 KO Suppresses Loss of Muscle Despite CKD or Type 1Diabetes

In gastrocnemius muscles of mice with CKD, the level of p-Stat3 wasincreased compared to results in muscles of pair-fed, sham-operated,control mice (FIG. 20A). To explore if the activation of Stat3 triggersmuscle wasting in vivo, mice with muscle-specific deletion of the Stat3tyrosine phosphorylation site (Stat3 KO) were studied, compared toresults in control, Stat3^(flox/flox) mice (Takeda et al., 1998). Micewith muscle-specific Stat3 KO did not differ from control mice in termsof development, food intake and body weight (FIG. 27). But with CKD,body weights of Stat3 KO mice increased vs. results in pair-fedStat3^(flox/flox) mice with CKD (FIG. 20B). The gain in weight was duein part to increased muscle mass: after 5 weeks of CKD, the weights ofgastrocnemius and tibialis anterior muscles were significantly greaterthan muscles from Stat3^(flox/flox) mice (FIGS. 20C, D). To determinewhy loss of muscle mass was blunted in Stat3 KO mice with CKD, rates ofmuscle protein synthesis and degradation were measured and there was asignificant improvement in both indices of protein metabolism in Stat3KO mice with CKD (FIGS. 20E, F). Likewise, there was an increase in gripstrength of Stat3 KO mice vs. Stat3^(flox/flox) mice (FIG. 20G).

Muscle atrophy in several catabolic conditions is characterized as anincrease in circulating inflammatory cytokines, impaired insulin/IGF-1signaling and an increase in muscle protein degradation via theubiquitin-proteasome system (UPS) (Zhang et al., 2011; Lecker et al.,2004). To determine if results present in mice with CKD occur in anothermodel of muscle wasting, streptozotocin-treated, acutely diabetic mice(Price et al., 1996) were studied. There was an increase in p-Stat3 plushigh circulating and muscle levels of IL-6 in acutely diabetic mice(FIG. 20H, FIG. 28). IL-6 mRNA in muscles of STZ-treated mice wasincreased 2-fold over control mice. Stat3 KO mice expressed a slowerdecrease in body weight vs. results in acutely diabetic,Stat3^(flox/flox) mice (FIG. 29). The slower loss of body weight inacutely diabetic Stat3 KO mice was associated with a greater mass ofgastrocnemius and tibialis anterior muscles vs. results inStat3^(flox/flox) mice (FIGS. 20, J). In the absence of CKD- ordiabetes-induced catabolism, muscle-specific Stat3 KO did notsignificantly affect body weight, muscle mass, protein metabolism orgrip strength compared to results in Stat3^(flox/flox) mice (FIGS.20A-20J). Thus, p-Stat3 can trigger muscle wasting in certain catabolicconditions.

Inhibition of Stat3 Activation Blocks CKD-Induced Muscle Wasting

To determine if a translational strategy might be developed to interferewith muscle wasting when Stat3 is activated, C188-9, a small moleculeinhibitor of Stat3 phosphorylation, was evaluated. C188-9 has a potencyin the low micromolar range and can be administered for prolongedperiods (Xu et al., 2009; Redell et al., 2011). After 2 weeks of CKD,mice were paired for their BUN and body weights and injected with eitherC188-9 or the diluent, 5% dextrose in water (D5W). C188-9 treatmentdecreased the level of p-Stat3 in muscle without affecting the Stat3level (FIG. 21A). Consistent with results from Stat3 KO mice with CKD,the body weights of CKD mice treated with the Stat3 inhibitor weresignificantly greater than weights of the control, CKD mice (FIG. 21B).After 14 days of C188-9, it was found that the increase in body weightincluded more muscle as the weights of gastrocnemius and tibialisanterior muscles were greater (FIGS. 21C, 21D). The increase in musclemass was confirmed by an analysis of the size distribution of myofibersin muscles of CKD mice treated with C188-9 (FIG. 21E). This improvementin muscle mass was accompanied by improved grip strength in CKD micetreated with C188-9 (FIG. 21F). Consistent with results from the Stat3KO mice, blocking Stat3 with C188-9 in control, wild type mice did notsignificantly affect their food intake, body weight, muscle mass or gripstrength (FIGS. 21B-D, F). The mechanism underlying the C188-9-inducedincrease in muscle weight included improved muscle protein synthesis anddecreased protein degradation (FIGS. 21G, H). Inhibiting Stat3activation suppresses CKD-induced loss of both muscle mass and strength.

In C2Cl2 Myotubes, Stat3 Activation Increases the Expression of C/EBPδand Myostatin

The signaling pathway from activated Stat3 to muscle wasting wasevaluated. Myostatin was studied because its expression is increased inmuscles of CKD mice and myostatin inhibition overcomes the decrease inprotein synthesis and the increase in protein degradation stimulated byCKD (Zhang et al., 2011). To determine how CKD leads to myostatinexpression, C/EBPδ was evaluated because the myostatin promoter hasseveral C/EBP recognition sites (Ma et al., 2001) and Stat3 can regulateC/EBPδ at least in epithelial cells (Zhang et al., 2007). First, C2Cl2myotubes were treated with IL-6 to activate Stat3. After 3 h, there wasan increase in the C/EBPδ protein in myotubes responding to activatedStat3. After 24 h, myostatin protein was increased and changes in mRNAswere consistent with the western blotting results (FIG. 22A, FIG. 30,31). These results show that p-Stat3, C/EBPδ and myostatin wereactivated sequentially.

Next, C2Cl2 myotubes were infected with a lentivirus which expresses aconstitutively active Stat3-GFP (Stat3C-GFP). The higher level ofp-Stat3 expression resulted in an increase in C/EBPδ and myostatin plusa decrease in p-Akt and myosin heavy chain (MHC) vs. results frommyotubes expressing GFP alone (FIG. 22B). Other evidence that Stat3activation stimulates myostatin expression was uncovered when theinhibitor of Stat3 (C188-9) was used to block p-Stat3 in C2Cl2 myotubes.After 24 h of exposure to IL-6, there was an increase in p-Stat3, C/EBPδand myostatin and C188-9 blocked these responses. The inhibitor alsoincreased p-Akt (FIG. 22C) and suppressed C/EBPδ and myostatin mRNAs inIL-6-treated C2Cl2 myotubes (FIG. 32). Notably, C188-9 not onlysuppressed p-Stat3 but also prevented the decrease in myotubes sizeinduced by exposure to IL-6 (FIG. 33).

To assess whether Stat3 affects C/EBPδ expression, C2Cl2 myoblasts wereco-transfected with a plasmid expressing a C/EBPδ promoter-drivenluciferase plus a lentivirus expressing the constitutively activeStat3C-GFP. Overexpression of Stat3C increased C/EBPδ promoter activitycompared to that in lentivirus expressing GFP control; addition of IL-6stimulated C/EBPδ promoter activity in myoblasts (FIG. 22D).

To identify whether p-Stat3 acts through C/EBPδ to stimulate myostatin,C/EBPδ was knocked down using siRNA. In this case, the IL-6-inducedincrease in myostatin expression was blocked when C/EBPδ was suppressedeven though p-Stat3 was increased (FIG. 22E). Next, C2Cl2 myoblasts wereco-transfected with a plasmid expressing myostatin promoter drivenluciferase plus one of the following: 1) a plasmid expressing Stat3C; 2)a plasmid expressing C/EBPδ; 3) C/EBPδ siRNA oligonucleotide; or 4) aplasmid expressing Stat3C and the C/EBPδ siRNA. Constitutively activeStat3C moderately increased myostatin promoter activity whiletransfection with C/EBPδ alone significantly increased myostatinpromoter activity. Knockdown of C/EBPδ blocked myostatin promoteractivity that was stimulated by IL6 or Stat3C (FIG. 22F).

C2Cl2 myoblasts were also transfected with a lentivirus that expressesmyostatin siRNA; it decreased myostatin expression and reduced proteindegradation even in cells expressing Stat3C or C/EBPδ (FIG. 22G, FIG.34). Thus, the Stat3 to C/EBPδ to myostatin pathway provides a mechanismcausing loss of muscle mass.

CKD-Induced Muscle Wasting In Vivo is Mediated by a Pathway from p-Stat3to C/EBPδ to Myostatin

In muscles of CKD or acutely diabetic mice, there were increases in theexpression of p-Stat3, C/EBPδ and myostatin (FIG. 23A, D). The C/EBPδand myostatin proteins in muscles of Stat3 KO mice with CKD weresignificantly below responses in muscles of Stat3^(flox/flox) mice withCKD. p-Smad2/3, the down stream signal of myostatin, expression was alsoincreased in muscles of CKD mice consistent with reports that p-Smad2/3mediates myostatin-induced muscle atrophy (Trendelenburg et al., 2009).The increase in p-Smad2/3 in muscle of mice with CKD was sharplydecreased in muscles of Stat3 KO mice with CKD. This suggests that inCKD, Stat3 activation results in myostatin expression and activation ofits downstream signaling pathway (FIG. 23A). Similar results were foundwhen C/EBPδ and myostatin mRNAs were examined in muscles of the Stat3 KOmice with CKD; levels in Stat3 KO mice with CKD were below those ofcontrol, Stat3^(flox/flox) mice with CKD (FIG. 23B, C). Activated Stat3in muscles of CKD mice was not completely blocked by muscle-specific KOof Stat3 when compared to p-Stat3 in muscles of non-CKD mice. Possibly,the remaining p-Stat3 in muscle lysates of Stat3 KO mice could reflectp-Stat3 in blood cells, blood vessels or the interstitium since theresults were obtained from western blots of gastrocnemius musclelysates.

When mice were treated with CKD using the inhibitor of Stat3, bothC/EBPδ and myostatin proteins were decreased and the CKD-inducedphosphorylation of p-Smad2/3 was blocked. In this case, the Aktphosphorylation was higher (FIG. 23E). Notably, C188-9 suppressed theCKD-stimulated mRNA expressions of C/EBPδ and myostatin (FIG. 23F, G).In control mice without CKD, muscle-specific Stat3 KO or C188-9treatment did not change either C/EBPδ or myostatin mRNAs or proteins inmuscle.

To demonstrate a link from Stat3 to C/EBPδ to myostatin in vivo, C/EBPδdeficient mice were studied that have normal embryonic development, arefertile and do not display overt developmental or physiological defects(Sterneck et al., 1998). CKD was created in heterozygous and homozygousC/EBPδ KO and wild type mice and fed the different groups the sameamount of chow as eaten by wild type mice with CKD. In homozygous C/EBPδKO mice with CKD, the loss of body and muscle weights were prevented.There also was improved survival in pair fed, homozygous C/EBPδ KO micewith CKD (FIGS. 24A-C). Despite the increase in p-Stat3 in muscles ofhomo- and heterozygous C/EBPδ KO or wild type mice with CKD, there wasno increase in expression of myostatin in mice with homozygous C/EBPδ KO(FIG. 24D). The degree of survival and myostatin expression in musclesof heterozygous C/EBPδ KO mice were intermediate between homozygous KOand wild type mice.

To examine whether Stat3-induced muscle wasting in vivo is mediated bymyostatin, a lentivirus expressing constitutively active Stat3-GFP(Stat3C-GFP) was injected into the right hindlimb of newborn mice. Theinjection was repeated 2 weeks later. At the same time, lentivirusexpressing GFP was injected into the left hindlimb (Control). Two weekslater, one group of mice was injected with anti-myostatin peptibody fortwo weeks; the other group was injected with PBS. Overexpression ofStat3C induced a significant reduction in myofiber sizes compared toresults in the contralateral hindlimb treated with the GFP. Notably,myostatin inhibition eliminated these responses. Next, muscle crosssections were immunostained with p-Smad2/3 and it was found that therewas high levels p-Smad2/3 in myofibers overexpressing Stat3C-GFP.Similar to results in CKD mice with muscle-specific Stat3 KO orfollowing treatment with the Stat3 inhibitor, the increase in p-Smad2/3was blocked by the anti-myostatin peptibody (FIGS. 24E, F, FIG. 35),consistent with a catabolic pathway from p-Stat3 to C/EBPδ tomyostatin-induced muscle protein loss.

In CKD Patients, there is Evidence for the p-Stat3, C/EBPδ to MyostatinPathway in Muscle

Muscle biopsies from patients with advanced CKD had significantlydecreased sizes of myofibers and levels of p-Akt (Table 12, FIG. 25A).There was, however, increased mRNA and protein levels of p-Stat3, C/EBPδand myostatin in muscles of CKD patients (FIGS. 25B-D).

Many catabolic conditions including CKD, diabetes, cancer and seriousinfections are complicated by progressive muscle wasting which decreasesthe quality of life and raises the risk of morbidity and mortality. Thecomplications of CKD (excess angiotensin II, glucocorticoids, acidosisand impaired insulin/IGF-1 signaling) stimulate protein degradation andloss of muscle mass. CKD also increases inflammatory markers includingIL-6, TNF-α and CRP et al which can activate p-Stat3 (Zhang et al.,2009; May et al., 1987; Hu et al., 2009; Zhang et al., 2011). Still, themolecular mechanisms causing muscle loss are poorly understood whichhampers the development of drug or other treatment strategies. In thepresent studies, it is identified that activated Stat3 triggers apathway from p-Stat3 to myostatin which causes the progressive musclewasting that is induced by CKD or acute diabetes.

Evidence for the p-Stat3-dependent pathway that initiates loss of musclemass was obtained in five experimental models: cultured C2Cl2 myotubes;muscle-specific p-Stat3 KO mice; mice treated with a small molecule thatinhibits Stat3 activation; C/EBPδ KO mice; and muscle biopsies ofpatients with CKD. The results show that CKD activates Stat3 leading toincreased expression of C/EBPδ and transcriptional regulation ofmyostatin expression. When this pathway is activated, there is adecrease in p-Akt which is shown will activate caspase-3 and theubiquitin-proteasome system (UPS) to degrade muscle protein (Zhang etal., 2011; Du et al., 2004; Wang et al., 2010). The results demonstratethat: 1) CKD or acute diabetes activates Stat3 in muscle causing loss ofmuscle mass; 2) targeted knockout of Stat3 in muscle or pharmacologicinhibition of Stat3 suppresses the muscle wasting that is induced by CKDor acute diabetes. This leads to an increase in muscle protein synthesisand a decrease in protein degradation with improvement in muscle massand grip strength; 3) C/EBPδ is a mediator of the pathway from p-Stat3to myostatin because its KO inhibits myostatin expression and suppressesmuscle wasting. In addition, C/EBPδ KO was associated with animprovement in survival. In muscle biopsies of patients with CKD, thereare similar changes in the levels of the same mediators suggesting theresults could form the basis for developing translation strategies tosuppress muscle wasting in CKD.

Presently, there are no clinically available drugs that directly targetStat3. Small molecule, drug development programs are initiated thattarget either the Stat3 homodimer interface or the Stat3 SH2 domain; thelatter is required for Stat3 binding to phosphotyrosylpeptide ligandslocated within activated receptor complexes and within the Stat3homodimer itself. Three candidate compounds (C3, C30 and C188) wereidentified; some identified single compounds including static (Schust etal., 2006), STA-21 (Song et al., 2005), S31-201 (Siddiquee et al., 2007)or LLL12 (Lin et al., 2010). Regarding LLL12, the evidence for directinhibition of Stat3 vs. an upstream kinase was not presented. Incontrast, C188-9, does not inhibit upstream JAK or Src kinases (Redellet al., 2011). Regarding potency, neither the parent compoundsidentified by others nor derivatives of them are as potent as C188-9(Bhasin et al., 2008; Zhang et al., 2010). In addition, compoundsidentified by others that have been tested in mice are not as welltolerated as C188-9 (Lin et al., 2009; Zhang et al., 2010); thosecompounds have a maximum tolerated dose of 5 mg/kg every 2 or 3 dayscompared to 100 mg/kg/day over 14 days for C188-9 (Tweardy et al,unpublished data). Thus, C188-9 has promise as a lead for developmentinto a drug that could be administered safely to patients.

How does C188-9 influence muscle protein wasting? One possibility isthat injection of IL-6 into rodents activates Stat3 and stimulatesmuscle proteolysis (Goodman, 1994). Indeed, there was increased IL-6 inmuscles of CKD patients and in STZ-induced acute diabetes in mice. Thelatter is consistent with reports from type 1 diabetic patients(Mysliwiec et al., 2006; Mysliwiec et al., 2008; Shelbaya et al., 2012).The potential origin of IL-6 in type 1 diabetes includes peripheralblood mononuclear cells and/or Th17 T cells (Bradshaw et al., 2009;Foss-Freitas et al., 2006; Ryba-Stanislawowska et al., 2013). However,others find that IL-6 does not stimulate muscle loss in the absence ofanother illness such as cancer (Baltgalvis et al., 2008). Thus, it isunclear how cytokines cause muscle proteolysis. In specific embodiments,the increase in IL-6 stimulated by CKD (Kimmel et al., 1998) andpossibly other cytokines, activates p-Stat3 which triggers musclewasting. Indeed, when Stat3 was deleted from muscle or when the Stat3inhibitor was studied, C188-9, CKD-induced muscle wasting was inhibited.How could p-Stat3 stimulate muscle wasting? p-Stat3 upregulates C/EBPδand increases the transcription of myostatin, a potent negativeregulator of muscle mass. Others have implicated C/EBPδ in thepathogenesis of catabolic disorders. For example, based on microarrayanalyses, there was upregulation of multiple genes including C/EBPδ inmuscles of mice with cancer cachexia or in muscle biopsies ofhemodialysis patients (Bonetto et al., 2011; Gutierrez et al., 2008). Inaddition, there are reports that p-Stat3 stimulates C/EBPδ expression incancer, immune or liver cells. This is relevant because the C/EBPδpromoter contains a Stat3 binding site making it a likely participant inthe pathway (Zhang et al., 2007; Sanford and DeWille, 2005). Indeed,exposure of C2Cl2 myotubes to IL-6 stimulates p-Stat3 and sequentiallyincreases the expression of C/EBPδ. Likewise, expression ofconstitutively active Stat3 in myotubes increased the C/EBPδ promoteractivity and the expression of the C/EBPδ protein. Contrariwise, Stat3inhibition in C2Cl2 myotubes or in CKD mice suppressed the expression ofC/EBPδ. A likely target of C/EBPδ is myostatin. For example, when thesiRNA to C/EBPδ was expressed in myotubes, the increase in myostatinstimulated by IL-6 was blocked. In addition, C/EBPδ KO in mice with CKDprevented their loss of muscle mass and expression of myostatin.Conclusions from these results are consistent with reports thatmyostatin is expressed in a wide variety of catabolic conditionsassociated with muscle wasting, including cancer, CKD, diabetes orweightlessness (spaceflight) (Zhou et al., 2010; Zhang et al., 2011;Feldman et al., 2006; Lalani et al., 2000). In mice with CKD, theactivation of Stat3 leads to expression of myostatin and its downstreamsignals, p-Smad2/3, plus accelerated protein degradation. Overexpressionof Stat3C in muscle of mice causes decreased myofiber sizes and knockingdown myostatin resolves the phenotype of Stat3 activation: myofibersizes are increased and p-Smad2/3 levels are reduced. Moreover,inhibition of myostatin suppresses the muscle wasting caused by CKD(Zhang et al., 2011). Finally, Zhou et al., reported that a soluble,actRIIB receptor inhibited myostatin and the UPS, blocking losses ofmuscle mass in several models of cancer (Zhou et al., 2010).

The mechanism by which an increase in myostatin leads to loss of musclemass could be a decrease in p-Akt in muscle. A decrease in p-Aktactivates caspase-3 leading to cleavage of the complex structure ofmuscle proteins and activation of proteolysis by the 26S proteasome (Duet al., 2004; Wang et al., 2010). In addition, a low p-Akt level wouldreduce phosphorylation of forkhead transcription factors which stimulatethe expression of the muscle-specific E3 ubiquitin ligases,Atrogin-1/MAFbx or MuRF-1 and accelerate proteolysis in the UPS (Sandriet al., 2004; Lee et al., 2004; Stitt et al., 2004; Lecker et al.,2006). In the present experiments, inhibition of p-Stat3 with C188-9decreased myostatin expression and the activation of its downstreamsignaling mediators, p-Smad2/3; there also was an increase in p-Akt. Inmuscle of CKD patients, as well, there was a sharp decrease in p-Aktwith increased mRNA and protein expressions of C/EBPδ and myostatin.

In summary, the results have uncovered a new pathway that stimulatesmuscle wasting in response to activation of Stat3. The pathway isactivated by CKD or acute diabetes and provides new insights into therelationships among the signaling molecules, Stat3, C/EBPδ, andmyostatin. Results from studies of cultured skeletal muscle cells ormice are consistent with changes in the levels of the same signalingmolecules in muscle biopsies of CKD patients. Consequently, theseresults are translatable into treatment strategies for catabolicconditions like CKD that causes muscle wasting. Development of a safeand potent small molecule Stat3 inhibitor is a useful therapeuticapproach to muscle wasting in catabolic conditions.

Experimental Procedures

Mouse Models

All animal experiments and procedures were approved by the BaylorCollege of Medicine Institutional Animal Care and Use Committee (IACUC).Subtotal nephrectomy was used to create CKD in mice (Zhang et al., 2011;May et al., 1987). To induce diabetes, 12-week-old Stat3^(flox/flox) andStat3 KO mice were injected intraperitoneally with 2 doses of 150mg/kg/d STZ (Sigma-Aldrich) in 0.1 M citrate buffer (pH 4). Control micewere injected with the citrate buffer. Mice were housed in individualcages and the diabetic Stat3^(flox/flox) mice were pair-fed withdiabetic Stat3 KO mice for 9 days.

Muscle Biopsies

During placement of a peritoneal dialysis catheter in CKD patients, therectus abdominis muscle was biopsied, frozen at −80° C. and stored untilanalyzed. Biopsy of this muscle was obtained from healthy subjectsduring abdominal hernia surgeries. The procedures were approved by theEthical Committee of the Department of Internal Medicine of theUniversity, Genoa, Italy, in accordance with the Helsinki declarationregarding ethics of human research.

mRNA Analyses

mRNAs were analyzed by RT-PCR as described (Takeda et al., 1998).Primers are listed in Table 13. Relative mRNA levels were calculatedfrom cycle threshold (Ct) values using GAPDH as the internal control[relative expression=2^((sampie Ct−GAPDH Ct))].

TABLE 13 Exemplary primer sequences for RT-PCR forward primersreverse primer Gene Accession (5′-3′) (5′-3′) C/EBP δ NM_005195TCTACATCTTACTCCTGT CAAATGCTGCTTTATTCT TGAT TACAA (SEQ ID NO: 1)(SEQ ID NO: 2) Myostatin NM_005259 CAACCTGAATCCAACTTA TGTTACCTTGACCTCTAA(SEQ ID NO: 3) (SEQ ID NO: 4) SOCS3 NM_003955 TTACAATCTGCCTCAATCATCTCCTAATAGCCTCAA (SEQ ID NO: 5) (SEQ ID NO: 6) GAPDH NM_002046CTCTGGTAAAGTGGATAT GGTGGAATCATATTGGAA TGT CA (SEQ ID NO: 7)(SEQ ID NO: 8) TNF-a NM_000594 CAACCTCTTCTGGCTCAA TGGTGGTCTTGTTGCTTA(SEQ ID NO: 9) (SEQ ID NO: 10) C/EBP 8 NM_007679 CTCCAGGGTCTAAATACACTCACAGCAGTCCACAAG   TAGC (SEQ ID NO: 12) (SEQ ID NO: 11) SOCS3NM_007707 CACAGCAAGTTTCCCGCC GTGCACCAGCTTGAGTAC GCC ACA (SEQ ID NO: 13)(SEQ ID NO: 14) Myostatin NM_010834 CTCCAGAATAGAAGCCATGCAGAAGTTGTCTTATAG A C (SEQ ID NO: 15) (SEQ ID NO: 16) Atrogin-1AF441120 GAGGCAGATTCGCAAGCG TCCAGGAGAGAATGTGGC TTTGAT AGTGTT(SEQ ID NO: 17) (SEQ ID NO: 18) MuRF-1 NM_001039048.2 AGTGTCCATGTCTGGAGGACTGGAGCACTCCTGCTT TCGTTT GTAGAT (SEQ ID NO: 19) (SEQ ID NO: 20) GAPDHNM008084 ACCACCATGGAGAAGGCC CTCAGTGTAGCCCAAGAT GG GC (SEQ ID NO: 21)(SEQ ID NO: 22)

Muscle Force Measurement

Mouse grip strength was measured daily for 4 consecutive days using aGrip Strength Meter (Columbus Instrument Co., Columbus, Ohio). Each day,5 grip strengths were assessed at 1 min intervals and the average gripstrength over 4 days was calculated.

Statistical Analysis

Data were expressed as the Mean±SEM. Differences between two groups wereanalyzed by the t test; multiple comparisons were analyzed by ANOVA witha post hoc analysis by the Student-Newman-Keuls test for multiplecomparisons. Results were considered statistically significant atp<0.05.

Reagents

Antibodies against p-Akt (Ser473), Akt, p-Stat3 (Tyr705), Stat3, P-Smad2(Ser465/467)/Smad3 (Ser423/425), p-IRS1 (Ser307) and IRS1 were from CellSignaling Technology (Beverly, Mass.); C/EBPδ were from ACRIS (SanDiego, Calif.); Smad3, myostatin, IL-6 and TNFα were from Abcam(Cambridge, Mass.); while those against laminin and MHC were fromSigma-Aldrich (St. Louis, Mo.). The GAPDH antibody was from Chemicon(Temecula, Calif.). IL-6 recombinant protein was from R&D Systems(Minneapolis, Minn.).

p-Stat3 Inhibitor

Three small-molecule probes (C3, C30 and C188) that target thephosphotyrosyl (pY) peptide binding site within the Stat3 SH2 domainwere identified using virtual ligand screening (Xu et al., 2009). Eachcompound competitively inhibited Stat3 binding to its pY-peptide ligandand ligand-induced Stat3 phosphorylation. C188 was the most potent ofthe three compounds identified. Similarity screening using thenaphthalenyl-benzenesulfamide scaffold of C188 followed by 3-Dpharmacophore analysis identified C188-9, which potently inhibited bothStat3 binding to its pY-peptide ligand (K_(i)=136 nM) and G-CSF-inducedStat3 phosphorylation (IC50=3±2 μM). Importantly, C188-9 at 10 μMconcentration did not inhibit upstream tyrosine kinases known toactivate Stat3, including Janus kinases (Jak1, Jak2) or Src familykinases (Hck, Lyn or Srms) as determined in a phosphoprotein array(RayBiotech, Norcross, Ga.) (Xu et al., 2009; Redell et al., 2011).

Cell Culture

C2Cl2 myoblasts (ATCC, Manassas, Va.) were grown in high glucoseDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 100U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml sodium pyruvate and 2mM L-glutamine. To obtain myotubes, myoblasts were cultured to 80-95%confluence and the media was changed to DMEM supplemented with 2% horseserum (Sigma-Aldrich). Myotubes were incubated in serum-free mediabefore being treated for different times with recombinant IL-6with/without the Stat3 inhibitor, C188-9. Myotube sizes were evaluatedby NIS-element software. Cell transfection was achieved byelectroporation with Amaxa Nucleofector (Lonza, Allendale, N.J.). C2Cl2myoblasts (106) were electroporated with 0.5 μg control or C/EBPδsiRNAs. Alternatively, 2 μg plasmids expressing Stat3C or C/EBPδ orC/EBPδ promoter- or myostatin promoter-reporter luciferase constructwere trasfected (control, Renilla) and luciferase activities wasmeasured using the Promega assay (Madison, Wis.).

Western Blotting

Muscle samples (1 mg per 10 μl of RIPA buffer) or C2Cl2 myotubes werehomogenized in RIPA buffer containing Complete Mini Protease Inhibitorand PhosStop Phosphatase Inhibitor (Roche Applied Science, Indianapolis,Ind.). Lysates were centrifuged for 5 min at 16,200×g at 4° C. and equalamounts of protein from the supernatant were separated onSDS-polyacrylamide gels in Tris/SDS buffer, transferred ontonitrocellulose membranes and incubated with primary antibodies overnightat 4° C. After washing with TBST, the membrane was incubated withsecondary antibodies conjugated to IRDye (Cell Signaling, Beverly,Mass.) at room temperature for 1 h. Protein bands were scanned using theOdyssey system (LI-COR, Lincoln, Nebr.). The band density of targetproteins was quantified using NIH ImageJ Software.

Immunohistochemical Staining

Cryo-sections (10 μm) of the midbelly region of tibialis anterior (TA)muscles were fixed in 4% paraformaldehyde and incubated withanti-laminin before exposing them to an Alexa Fluor 488-conjugated mouseIgG secondary antibody (Invitrogen, Grand Island, N.Y.). Nuclei werestained with DAPI. Myofiber sizes were measured using NIS-Elements Br3.0 software (Nikon) and the size distribution was calculated from 2000myofibers by observers blinded to treatments. Paraffin sections (5 mm)of human muscles were immunohistochemically stained for the expressionof p-Stat3, IL-6 and TNFα by incubating them with the primary antibodyfor 1 h at room temperature followed by incubation for 30 min withbiotinylated antibodies. IL-6 and TNFα expression in 3 representativeareas of the muscle sections was analyzed and expressed as thepercentage of the myofiber area that stained positively. Stat3 in nucleiwas expressed as the percent of nuclei positive for p-Stat3 in a totalof 550 nuclei; the observer was blinded to patient vs. healthy subject.

Mouse Models

Transgenic mice expressing muscle creatine kinase-Cre (Mck-Cre) fromJackson Laboratory (Bar Harbor, Me.) were cross-bred with Stat3flox/floxmice with loxP sites flanking portions of exons 21 and 22 of the Stat3gene. This site encodes a tyrosine residue (Tyr705) that is essentialfor Stat3 activation (Takeda et al., 1998). Mice expressing both Mck-Creand Stat3flox/flox (i.e., Stat3 KO) were identified by genotyping andWestern blotting. Heterozygous, C/EBPδ deficient mice were a gift fromDr. E. Sterneck (NIH—NCI, Frederick, Md.). Homozygous, C/EBPδ KO micewere developed by cross breeding of C/EBPδ heterozygous mice andPCR-genotyping. Subtotal nephrectomy was used to create CKD in wildtype, Stat3flox/flox, Stat3 KO and C/EBPδ deficient mice (Zhang et al.,2011; May et al., 1987). Briefly, anesthetized mice underwent subtotalnephrectomy in two stages followed by a weeklong recovery while theywere eating a 6% protein diet to reduce mortality from uremia.Subsequently, uremia was induced by feeding these and control mice a 40%protein diet. Mice were housed in 12-h light-dark cycles and bodyweights and food eaten were assessed daily. The influence of the Stat3inhibitor, C188-9, was tested in CKD mice paired for BUN, body weightsand chow intake; one mouse was injected subcutaneously with 6.25 mg/kgof C188-9 in D5W daily for 14 days while the paired CKD mouse wasinjected with an equal amount of D5W. Stat3 KO mice with CKD were fedthe same amount of chow as the wild type mice with CKD. Homozygous andheterozygous C/EBPδ KO mice with CKD were fed the same amount of food aseaten by wild type mice with CKD for 14 days.

Lentivirus Production and Transfection

To produce lentiviruses to express Stat3C-GFP or GFP, 5×106 293T cellswere cotransfected with 2 μg EF.STAT3C.Ubc.GFP or GFP (AddgeneCambridge, Mass.) plus 1 μg HIV-1 packaging vector 68.1 plus 0.4 μg VSVGenvelope using Lipofectamine 2000. After 48 h, the virus pellet wascollected by centrifugation (50,000×g for 2 h), re-suspended inTris-NaCl-EDTA buffer and stored at −80° C. C2Cl2 myotubes weretransfected with 107 virus particles/ml of DMEM plus 10% FBS and 5 μg/mlpolybrene and 48 h later, proteins were evaluated by western blot.

For in vivo transfection, 10 μl of 10⁷ virus particles/ml of Stat3C-GFPwas slowly injected into right hindlimb of newborn C57/BL6 mice; GFP wasinjected into the left hindlimb as a control. Two weeks later, thelentivirus injections were repeated. Mice were divided into twogroups: 1) anti-myostatin peptibody treatment for two weeks as described(Zhang et al., 2011); or 2) an equal volume of PBS. At 6 weeks after theinitial injection, sizes of myofibers expressing GFP were measured.Myostatin- and control-shRNAs lentivirus particles were from Santa CruzTechnology; ˜50% confluent C2Cl2 myoblasts were transfected with 105virus units in 8 μg/ml polybrene and selected by 5 μg/ml puromycin.Selected clones were tranfected with Stat3C or C/EBPδ and used tomeasure protein degradation after differentiation into myotubes.

Protein Synthesis and Degradation

Extensor digitorum longus (EDL) muscles were maintained at restinglength and incubated in Krebs-Henseleit bicarbonate buffer with 10 mMglucose as described (Zhang et al., 2011). L-[U-¹⁴C] phenylalanineincorporation into muscle protein and tyrosine release were measured asrates of protein synthesis and degradation (Clark and Mitch, 1983). Incultured C2Cl2 myotubes treated to knockdown myostatin or overexpressStat3C or C/EBPδ, protein degradation in cells prelabeled with L-[U-¹⁴C]phenylalanine was calculated from radiolabeled phenylalanine release(Zhang et al., 2009). The measurements were repeated six times.

Example 12 Inhibiting Stat3 Activation Suppresses Cancer-Induced MuscleWasting

As described in this example, while evaluating cachexia it was foundthat conditioned media from C26 colon carcinoma or Lewis lung carcinoma(LLC) cells, activated p-Stat3 in C2Cl2 myotubes, followed by expressionof C/EBPδ and myostatin with reduced myotube mass. In mice, LLC causedmuscle wasting via a pathway from p-Stat3 to C/EBPδ to myostatin andactivation of proteolysis by caspase-3 and the ubiquitin-proteasomesystem. Muscle-specific Stat3 KO suppressed cancer cachexia withoutreducing tumor growth. In mice with LLC, C/EBPδ KO blocked myostatin andthe loss of body and muscle weights with improved grip strength. Sincep-Stat3 initiates muscle wasting, it was evaluated whether a smallmolecule inhibitor of p-Stat3, C188-9, blocks cancer cachexia. In micewith C26 cancer, C188-9 blocked Stat3 activation, increased body andmuscle weights while improving grip strength. C188-9 improved thesynthesis and degradation of muscle proteins resulting in increasedmyofiber sizes. Thus, p-Stat3 inhibition genetically or chemicallysuppresses a pathway causing muscle wasting in these cancer models.Blocking the pathway could lead to novel therapeutic strategies toprevent cancer-induced muscle atrophy.

Material and Methods

Animals

All animal experiments and procedures were approved by the BaylorCollege of Medicine Institutional Animal Care and Use Committee (IACUC).CD2F1 mice (Charles River; Houston, Tex.) were studied at 8-10 weeks ofage following subcutaneous injection of C26 tumor cells (5×10⁶ cells in500 μL medium) in the right flank. After 5 days, tumor bearing mice weretreated with daily injections of the diluent, D5W (control), or C188-9(12.5 mg Stat3 inhibitor/kg body weight). Control and cancer bearingmice were pair-fed for 14 days and growth was measured. Forpair-feeding, the amount eaten by the cancer-bearing mouse was fed tothe paired, control mouse the following day.

Mice were studied with muscle-specific knockout of Stat3 (Stat3 KO) orC/EBPδ KO mice. Stat3 KO mice were created by breeding transgenic miceexpressing Stat3^(flox/flox) with mice expressing muscle creatine kinaseCre (MCK-Cre) (Zhang et al., 2013). The Stat3 KO or C/EBPδ KO mice wereimplanted with 5×10⁶ LLC cancer cells and body weights were measuredduring 12-14 days of pair feeding. Subsequently, mixed fiber tibialisanterior (TA) and gastrocnemius muscles, the predominately red myofiber,soleus muscle, and the white myofiber extensor digitorum longus (EDL)muscles were dissected, weighed, immediately frozen in liquid nitrogenand stored at −80° C.

Reagents

Antibodies against total-Stat3 and phospho-Stat3 were purchased fromCell Signaling Technology (Beverly, Mass.). Antibodies against C/EBPδwere from Acris Antibodies (San Diego, Calif.), against Atrogin1/MAFbxand MuRF1 from Santa Cruz Biotechnology (Santa Cruz, Calif.), againstmyostatin from Abcam (Cambridge, Mass.) and againstglyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Milipore(Temecula, Calif.).

Cell Culture Studies

Mouse C2Cl2 myoblasts (ATCC, Manassas, Va.) and LLC cells (Dr. Yi-PingLi; University of Texas Health Sciences Center, Houston, Tex.) werecultured in DMEM (Cellgro Mediatech, Manassas, Va.), supplemented with10% FBS (Invitrogen, Carlsbad, Calif.) plus 100 U/ml penicillin and 100g/ml streptomycin. C26 cells (a gift from Dr. Vickie Baracos, Universityof Alberta, Edmonton, Alberta, Canada) were cultured in RPMI 1640 medium(Sigma-Aldrich), supplemented with 10% FBS (Invitrogen, Carlsbad,Calif.), 100 U/ml penicillin, and 100 g/ml streptomycin.

At >80% confluence, the media was changed to DMEM supplemented with 2%horse serum (Sigma-Aldrich) to induce myoblasts to differentiate intomyotubes (Zhang et al., 2011). After 36 h, conditioned media (CM) fromcultured C26 or LLC cells was collected and centrifuged (450×g, 5 min,4° C.); media was diluted 1:5 with 2% horse serum before adding culturedC2Cl2 myotubes (Zhang et al., 2011).

Real-Time PCR

RNA from gastrocnemius muscles was obtained using RNeasy (Qiagen,Valencia, Calif.). cDNAs were synthesized using the iScript advancedcDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif.). Real-timePCR was performed with a CFX96 RT-PCR machine and SYBR Green (Bio-RadLaboratories). The relative mRNA expression levels were calculated fromcycle threshold (Ct) values using glyceraldehyde 3-phosphatedehydrogenase (GAPDH) as the internal control (relativeexpression=2^((sample Ct−GAPDH Ct))) Primer sequences have beendescribed (Zhang et al., 2013).

Protein Synthesis and Degradation

As described Soleus and EDL muscles were rapidly removed from mice(Zhang et al., 2011; Clark and Mitch, 1983). The muscles were maintainedat resting length during incubation in Krebs-Henseleit bicarbonatebuffer with 10 mM glucose and L-[U-¹⁴C]phenylalanine. Protein synthesiswas measured as the rate of incorporation of L-[U-¹⁴C]phenylalanine inmuscle protein while protein degradation was assessed as the release oftyrosine from muscle proteins undergoing degradation. Phenylalanine andtyrosine were studied because they are neither synthesized nor degradedby muscle and they rapidly equilibrate with the intracellular free aminoacid pool in muscle.

Caspase-3 Promoter Assay

Using the MI Inspector program, 3 putative STAT3 binding sites wereidentified in the caspase-3 promoter. Dr. Sabbagh (Montreal, Quebec,Canada) kindly provided us with a series of deletions of the caspase-3promoter in a luciferase reporter construct. Different constructscontained zero, 1,2 or 3 putative Stat3 binding sites while a reversecaspase-3 promoter sequence was used as a negative control. Eachcaspase-3 promoter-luciferase reporter construct was electroporated intoC2Cl2 cells. Cells containing the different constructs were treated by100 ng/ml IL-6 or a plasmid expressing constitutively active Stat3 orboth. Luciferase activity in these cells was compared to luciferaseactivity in cells incubated in serum free media. Luciferase activity incell lysates was measured using the dual-luciferase reporter-assaysystem.

ChIP Assay:

C2Cl2 myoblasts were infected with an adenovirus expressing Stat3 andthen treated with or without IL-6. Myoblasts were crosslinked with 1%formadehyde for 15 min at RT and washed 3× with ice-cold PBS containinga protease inhibitor (Roche). Myoblasts were lysed in lysis buffer,vortexed and sonicated for 10 sec at power setting 4; this was repeated4× (VibraCell Sonicator). The average lengths of DNA fragments rangedbetween 300 and 800 bp. After centrifugation, the protein-DNA lysate wasdiluted 10-fold in ChIP buffer (15 mM Tris (pH 8.0), 1% Triton X-100,0.01% SDS, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1/100 proteaseinhibitor cocktail). the samples were precleared using salmon sperm DNAand protein A/G Agarose beads for 1 h at 4° C. Each 100 μL ofprotein-DNA lysate was used as an input control.

Samples were immunoprecipitated with antibodies to Stat3, p-Stat3 orRabbit IgG (Santa Cruz Biotechnology) overnight at 4° C. followed byincubation with protein A/G Agarose beads for 1 h at 4° C. The immunecomplexes were washed as described by the manufacturer.Immunoprecipitated DNA was reverse crosslinked at 65° C. for 4 h in thepresence of 0.2 M NaCl and purified usingphenol/chloroform/isoamylalcohol. A total of 5 μl of the purified DNAwas subjected to PCR amplification of a 190-bp fragment using specificprimers that were derived from the promoter region of the caspase-3gene.

Proteasome Activity

Proteasomes were partially purified by differential centrifugation;equal amounts of protein from the preparations of proteasomes were usedto measure proteasome activity as the release of7-amino-4-methylcoumarin (AMC) from the fluorogenic peptide substrateLLVY-AMC (N-Suc-Leu-Leu-Val-Tyr-AMC). AMC fluorescence was measuredusing 380 nm excitation and 460 nm emission wavelengths. The differencebetween the fluorescence measured in the presence and absence of 100 μmlactacystin was used to calculate proteasome activity.

Muscle Force

Mouse grip strength was measured as described (Zhang et al., 2013).Briefly, 5 grip strengths were assessed at 1 min intervals using theGrip Strength Meter (Columbus Instrument Co., Columbus, Ohio). Theaverage grip strength over 4 days was calculated.

Statistical Analysis

Student's t test was used when 2 experimental groups were compared andANOVA when data from 3 or 4 groups were studied. After ANOVA analyses,pairwise comparisons were made by the Student-Newman-Keuls test. Thedata are presented as means±SEM.

Conditioned Media from Cultured C26 or LLC Cancer Cells Stimulates C2Cl2Myotube Atrophy Via a Pathway from p-Stat3 to C/EBPδ to Myostatin.

The presence of cancer cachexia in patients or rodent models suggeststhat cancer cells release a factor(s) that stimulates the loss of musclemass (Todorov et al., 1996). In exploring potential mediators ofcachexia, conditioned media was added from cultures of C26 to C2Cl2myotubes. Within 5 minutes, the conditioned media stimulated myotuberesponses that included a >10-fold increase in activated(phosphorylated) Stat3 (p-Stat3) (FIG. 36A). However, when the Stat3inhibitor C188-9 was added to C2Cl2 myotubes 2 h before adding the C26or LLC conditioned media, the increases in p-Stat3 (15 min forconditioned media) were blocked (FIG. 36B). The conditioned media alsoincreased the expressions of C/EBPδ and myostatin in cultured C2Cl2myotubes and they were suppressed by C188-9 (FIG. 36C). These resultsare relevant to the development of cachexia because the conditionedmedia reduced the sizes of myotubes and treatment with the Stat3inhibitor, C188-9, prevented the decrease in sizes of myotubes (FIG.36D).

Muscle-Specific Stat3 KO in Mice with LLC Tumors Improves SkeletalMuscle Metabolism

The finding that media from cultured cancer cells activates Stat3,increases the expression of C/EBPδ and myostatin and causes atrophy ofmyotubes suggests that C/EBPδ and myostatin are “downstream” from Stat3activation (FIG. 36) (Zhang et al., 2013). Because the increases inC/EBPδ and myostatin levels in C2Cl2 myotubes are suppressed by theC188-9 inhibitor of p-Stat3, in another embodiment it would be thatC188-9 is not specific but also inhibits C/EBPδ and myostatin resultingin improvements in muscle metabolism (Zhang et al., 2013). To evaluatethe latter consideration, mice were generated with muscle-specific Stat3KO. These Stat3 KO mice are fertile and develop normally (Zhang et al.,2013). These KO mice and control, Stat3^(flox/flox) mice were injectedsubcutaneously with LLC and then pairfed for 18 days. During thepair-feeding, the genetically altered mice were injected with LLC andgiven the same amount of food as that eaten on the prior day by the LCCtumor bearing, Stat3^(flox/flox) mice. In the Stat3^(flox/flox) mice,the LLC tumor caused a significant decrease in the body weight (FIG.37A). In contrast, mice with muscle-specific, Stat3 KO had animprovement in their growth, reaching a level that was indistinguishablefrom that of control mice without the LLC cancer (FIG. 37A). Stat3 KObearing tumor had higher body weight maybe due to increased amount ofmuscle mass vs. control mice bearing tumor (FIG. 37C). When the micewere examined in the absence of the LLC tumor, it was found that micewith muscle-specific Stat3 KO mice had the same growth asStat3^(flox/flox) mice. This result indicates that muscle-specific KO ofStat3 did not interfere with the growth of the genetically alteredmouse. In addition, the responses were independent of changes in tumormass (FIG. 37B). Both C/EBPδ and myostatin expressions in mice withmuscle-specific Stat3 KO, were decreased similarly to the responsesnoted when the Stat3 inhibitor, C188-9, was added to C2Cl2 myotubesbeing treated with conditioned media from cancer cells (FIGS. 37D; 36C).in consistent with increased muscle mass, Stat3 KO mice bearing tumorshowed higher muscle grip strength vs. constol Stat3^(flox/flox) bearingtumor (FIG. 37E). Thus, in vivo genetic inhibition of p-Stat3 producesresults like those achieved by suppressing p-Stat3 with C188-9 in vitro,consistent with the conclusion that C188-9 functions as an inhibitor ofp-Stat3.

C/EBPδ KO in Mice Suppresses LLC Tumor-Induced Cachexia

Because LLC or C26 cancers increase the expressions of p-Stat3, C/EBPδplus myostatin in muscle plus inhibition of myostatin blocks musclewasting (Han et al., 2013), it was examined whether C/EBPδ also isnecessary for the muscle wasting that follows activation of p-Stat3.Mice with whole body, homozygous C/EBPδ KO were created from C57BL6mice; the mice are fertile and develop normally and they respondadversely to cancer. When LLC cancer cells were injected subcutaneouslyin the genetically altered and control mice, the absence of C/EBPδ didnot affect the growth of LLC tumors. The weights of body and muscles ofC/EBPδ KO mice were preserved but in WT mice bearing LLC, there was lossof body and muscle weights (FIGS. 38A, B). Notably, LLC caused adecrease in weights of the different types of muscles including themixed-fiber gastrocnemius and tibialis anterior muscles as well as thered-fiber (soleus) and white fiber (extensor digitorum longus) muscles.

A mechanism that contributes to the loss of muscle mass in WT mice withLLC is an increase in protein degradation in muscles. This response wassignificantly reduced in C/EBPδ KO mice (FIG. 38C). The improvements inmuscle mass and metabolism were associated with an increase in gripstrength in C/EBPδ KO mice (FIG. 38D). Consistent with the proposedsignaling pathway, C/EBPδ KO suppressed tumor induced myostatin level inmuscle of mice (FIG. 38E). Taken together, the results demonstrate thatC/EBPδ is required for the pathway that links p-Stat3 to loss of musclemass.

Inhibition of Stat3 Activation Improves Cancer-Induced Muscle Wasting

To examine potential mechanisms causing cancer-induced loss of musclemass, C26 cancer cells were injected into CD2F1 mice. As reported byothers (Aulino et al., 2010), implantation of C26 tumors in mice causesa pronounced loss of body weights (FIG. 39B). Besides loss of musclemass, there was a significant increase in pStat3 in muscles of micebearing C26 tumors (FIG. 39A). To investigate whether body weight lossdepended on Stat3 activation, mice bearing C26-tumor cells were treatedwith a small molecule inhibitor of p-Stat3, C188-9, for 14 daysbeginning at 5 days after tumor implantation. In tumor-bearing micetreated with C188-9, Stat3 activation was suppressed.

There also was a significant increase in body weight even though the C26tumor had been in place for 19 days. The ability of C188-9 to improvebody weight included blockade of muscle wasting since the weights of themixed fiber tibialis anterior (TA) and gastrocnemius muscles as well asthe predominately red fiber soleus and white fiber EDL muscles weresignificantly greater than the weights of pair-fed, tumor bearing micethat were treated with D5W (FIG. 39C). The myofiber sizes are consistentwith muscle mass (FIGS. 39D&E). Notably, the increase in muscle mass intumor bearing mice led to improved grip strength, a measure of musclefunction (FIG. 37G). The mechanisms underlying for improvements inmuscle mass included an increase in protein synthesis plus a decrease inprotein degradation resulting in an improvement in the sizes ofmyofibers (FIGS. 39F&G). There was an increased rate of proteinsynthesis and degradation in both white (EDL) and red (soleus) fibers.

p-Stat3 Stimulates the Transcription of Caspase-3, Participating in theDevelopment of Cancer Cachexia.

Caspase-3 is an initial step for muscle wasting, because caspase-3 playstwo roles in promoting muscle proteolysis: first, caspase-3 cleaves thecomplex structure of muscle proteins to provide substrates for the UPS(Du et al., 2004; Song et al., 2005; Zhang et al., 2009). Second,caspase-3 cleaves specific subunits of the 19S proteasome particle thatstimulates proteolytic activity of the 26S proteasome (Wang et al.,2010). Besides these properties, caspase-3 activation can be recognizedby the presence of a 14 kD actin fragment that is left in the insolublefraction of muscle biopsies (Du et al., 2004; Workeneh et al., 2006). Toevaluate whether there is tumor induce caspase-3 expression andactivation in muscle mass loss, procaspase-3 and cleaved caspase-3 weremeasured in muscle of mice bearing C26 or LLC tumor; both tumorsstimulated pro-caspase-3 and cleaved caspase-3 level in muscle (FIG.40A). The caspase-3 activity also increased, because in muscle of micebearing either tumor, there was an increased 14 kD actin fragment (FIG.40B).

Next, it was measured whether the increased caspase-3 expression islinked to tumor induced p-Stat3 in muscle. Using MatInspector program,there were three putative Stat3 binding sites in the 3 kb promoterregion of caspse-3. To test if cancer cell media induced p-Stat3stimulate it binding with caspase-3 promoter, C2Cl2 cells were treatedwith C26 conditioned media for 24 h and subjected to ChIP assay usinganti-p-Stat3, the DNA associated with p-Stat3 was amplified usingprimers from caspase-3 promoter, there was DNA fragment from PCRamplification in C2Cl2 myotubes treated with C26 conditined media, butnot in control cells (C2Cl2 myotubes in serum free media) (FIG. 40C). Tofurther determine that Stat3 could binding with caspase-3 promoter tostimulate caspase-3 transcription, C2Cl2 myotubes were infected withadenovirus expressing Stat3. The control cells infected adenovirusexpressing GFP. These cells treated with or without IL-6 (100 ng/ml) for24 h. CHIP assay using anti-Stat3 indicate that cells without eitheroverexpressing Stat3 or IL-6 treatment does not show Stat3 binding withcaspase-3. Stat3 do binding with caspase-3 in cells overexpressing Stat3and treated with IL-6. P-Stat3 binding with caspase-3 in any donsitionsand there was a strong interaction between caspase-3 and p-Stat3 incells onverexpressing Stat3 and stimulated with IL-6 (FIG. 40D). To testwhether p-Stat3 binding with caspse-3 promoter to stimulates caspase-3transcription, C2Cl2 myoblast was transfected with plasmid of caspase-3promoter in luciferase construct and plasmid to express constitutivelyactive Stat3 (Stat3C). Control cells transfected with cDNA3. Cells weretreated with or without IL-6 for 6 h. There was IL-6 or StatC stimulatedcaspase-3 promoter activity, but cells stimulated both IL-6 andoverexpressing Stat3 stimulated the highest caspase-3 promoter activity.When all three Stat3 binding site were deleted in caspase-3 promoter(−178/+14), there is no stimulated caspase-3 promoter activity foundeven with IL-6 or Stat3C or both (FIG. 40E). These results indicate thatp-Stat3 binding with caspase-3 to stimulate its expression.

Activation of Stat3 Induces Ubiquitin-Proteasome System inCancer-Induced Cachexia.

When C2Cl2 myotubes were treated with conditioned media from C26 cellswith or without C188-9 for 72 hours, there was decreased protein levelof myosin heavy chain, and this response is blocked by C188-9 (FIG.41A). To test if this proteolysis involved UPS, Atrogin and MuRF-1 mRNAexpression were measured in these cells; there was significantlyincreased expression of both muscle specific ubiquitin E3 lygases, andthis response is suppressed by C188-9 (FIG. 41A). To test whether thisis the case in muscle of mice, Atrogin-1 and MuRF-1 expression levelswere measured in Stat3 KO or C188-9 treatment muscle of mice bearingtumor, and these results are consistent with results in cell culture.Finally, there was increased proteasome activity in muscle of micebearing C26 tumor and it was suppressed by C188-9 (FIG. 41D). Therefore,Stat3 activation occurs in muscle of mice bearing tumor stimulated UPSto induce muscle wasting.

As an example, FIG. 42 exemplifies how in one embodiment cancer thatactivates p-Stat3 in muscle can stimulate loss of muscle mass. Stat3activation stimulates expression of C/EBPδ that then increases myostatinand MAFbx/Atrogin-1 and MuRF-1 to increase muscle wasting by the UPS.Stat3 activation also increases caspase-3 expression and activity tocoordinate muscle proteolysis with the UPS.

REFERENCES

All patents and publications cited herein are hereby incorporated byreference in their entirety herein. Full citations for the referencescited herein are provided in the following list.

PUBLICATIONS

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What is claimed is:
 1. A method of treating muscle wasting, muscleweakness, or cachexia, or a combination thereof, in an individual inneed thereof, comprising administering to the individual an effectiveamount of a compound selected from the group consisting of N-(1‘,2-dihydroxy-1,2’-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide,4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide,and4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide,or a pharmaceutically acceptable salt thereof, wherein the musclewasting, muscle weakness, or cachexia is not associated with cancer. 2.The method of claim 1, wherein muscle wasting or muscle weakness isassociated with cachexia.
 3. The method of claim 1, wherein the musclewasting, muscle weakness, or cachexia, or a combination thereof, isassociated with an underlying medical condition.
 4. The method of claim3, wherein the underlying medical condition is chronic.
 5. The method ofclaim 3, wherein the underlying medical condition is selected from thegroup consisting of chronic kidney disease, diabetes, renal failure,AIDS, HIV infection, chronic obstructive lung disease, multiplesclerosis, congestive heart failure, tuberculosis, familial amyloidpolyneuropathy, acrodynia, hormonal deficiency, metabolic acidosis,infectious disease, chronic pancreatitis, autoimmune disorder, celiacdisease, Crohn's disease, electrolyte imbalance, Addison's disease,sepsis, bums, trauma, fever, long bone fracture, hyperthyroidism,prolonged steroid therapy, surgery, bone marrow transplant, atypicalpneumonia, brucellosis, endocarditis, Hepatitis B, lung abscess,mastocytosis, paraneoplastic syndrome, polyarteritis nodosa,sarcoidosis, systemic lupus erythematosus, visceral leishmaniasis,prolonged bed rest, and drug addiction.
 6. The method of claim 3,wherein the individual is administered a therapy for the underlyingmedical condition.
 7. The method of claim 3, wherein the individual isadministered an additional therapy for muscle wasting, muscle weakness,or cachexia, or a combination thereof, and the individual isadministered a therapy for the underlying medical condition.
 8. Themethod of claim 3, further comprising the step of diagnosing theunderlying medical condition.
 9. The method of claim 1, wherein theindividual is administered the compound, or a pharmaceuticallyacceptable salt thereof, in multiple doses.
 10. The method of claim 9,wherein the multiple doses are separated by hours, days, or weeks. 11.The method of claim 1, wherein the individual is administered anadditional therapy for muscle wasting, muscle weakness, or cachexia, ora combination thereof.
 12. The method of claim 1, wherein the compoundisN-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide,or a pharmaceutically acceptable salt thereof.
 13. The method of claim1, wherein the compound inhibits signal transducer and activator oftranscription 3 (Stat3), signal transducer and activator oftranscription 1 (Stat1), or both.
 14. The method of claim 1, furthercomprising the step of diagnosing muscle wasting, muscle weakness, orcachexia, or a combination thereof.
 15. The method of claim 1, whereinthe compound is delivered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularly, orally, topically, locally, injection,infusion, continuous infusion, localized perfusion, via a catheter, viaa lavage, in lipid compositions, in liposome compositions, or as anaerosol.